U.S. patent application number 13/703123 was filed with the patent office on 2013-10-10 for system and method of modular cloning.
This patent application is currently assigned to ICON GENETICS GMBH. The applicant listed for this patent is Carola Engler, Ramona Grutzner, Sylvestre Marillonnet, Ernst Weber, Stefan Werner. Invention is credited to Carola Engler, Ramona Grutzner, Sylvestre Marillonnet, Ernst Weber, Stefan Werner.
Application Number | 20130267021 13/703123 |
Document ID | / |
Family ID | 43242437 |
Filed Date | 2013-10-10 |
United States Patent
Application |
20130267021 |
Kind Code |
A1 |
Weber; Ernst ; et
al. |
October 10, 2013 |
SYSTEM AND METHOD OF MODULAR CLONING
Abstract
System for producing a nucleic acid construct of interest, said
system comprising: a set of n entry DNAs numbered 1 to n, n being
an integer of at least 2, each of said n entry DNAs comprising in
this order: (i) a type IIs restriction endonuclease recognition
site followed by the cleavage site thereof; (ii) a sequence portion
linking the cleavage site of said recognition site of item (i) with
the cleavage site of the recognition site of the following item
(iii), and (iii) a cleavage site of a further type Ms restriction
endonuclease recognition site followed by the recognition site of
said cleavage site; the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (iii) of entry DNAs 1 to n-1
are complementary to the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (i) of entry DNAs 2 to n,
respectively; the cleavage site of the type Ms restriction
endonuclease recognition site of item (iii) of entry DNA n is
complementary to the cleavage site of the type IIs restriction
endonuclease recognition site of item (i) of entry DNA 1 for
allowing annealing of complementary single-stranded overhangs
formed by restriction at recognition site (i) of entry DNA 1 and at
recognition site (iii) of entry DNA n; said system further
comprising a destination vector comprising in this order: (I) a
type Ms restriction endonuclease recognition site followed by the
cleavage site thereof; (II) a vector backbone preferably comprising
a selectable marker gene, said vector backbone linking the cleavage
sites of said recognition sites of items (I) and the following item
(III); (III) a further cleavage site of a type Ms restriction
endonuclease recognition site followed by the recognition site of
said cleavage site, and (IV) optionally, an insert between the
recognition sites of item (III) and item (i); said cleavage sites
of items (I) and (III) being different and non-complementary, said
recognition sites of items (I) and (III) being preferably
recognitions sites of the same endonuclease.
Inventors: |
Weber; Ernst; (Halle/Saale,
DE) ; Werner; Stefan; (Halle/Saale, DE) ;
Engler; Carola; (Halle/Saale, DE) ; Grutzner;
Ramona; (Halle/Saale, DE) ; Marillonnet;
Sylvestre; (Halle/Saale, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Weber; Ernst
Werner; Stefan
Engler; Carola
Grutzner; Ramona
Marillonnet; Sylvestre |
Halle/Saale
Halle/Saale
Halle/Saale
Halle/Saale
Halle/Saale |
|
DE
DE
DE
DE
DE |
|
|
Assignee: |
ICON GENETICS GMBH
Munchen
DE
|
Family ID: |
43242437 |
Appl. No.: |
13/703123 |
Filed: |
June 9, 2011 |
PCT Filed: |
June 9, 2011 |
PCT NO: |
PCT/EP2011/002843 |
371 Date: |
May 14, 2013 |
Current U.S.
Class: |
435/320.1 |
Current CPC
Class: |
C12N 15/1093 20130101;
C12N 15/66 20130101 |
Class at
Publication: |
435/320.1 |
International
Class: |
C12N 15/66 20060101
C12N015/66 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 11, 2010 |
EP |
10 006 090.4 |
Jul 6, 2010 |
EP |
10 006 955.8 |
Claims
1. System for producing a nucleic acid construct of interest, said
system comprising: a set of n entry DNAs numbered 1 to n, n being
an integer of at least 2, preferably at least 3, each of said n
entry DNAs comprising in this order: (i) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; (ii) a sequence portion linking the cleavage site of said
recognition site of item (i) with the cleavage site of the
recognition site of the following item (iii), and (iii) a cleavage
site of a further type IIs restriction endonuclease recognition
site followed by the recognition site of said cleavage site; the
cleavage sites of the type IIs restriction endonuclease recognition
site(s) of item (iii) of entry DNA(s) 1 to n-1 is/are complementary
to the cleavage site(s) of the type IIs restriction endonuclease
recognition site(s) of item (i) of entry DNA(s) 2 to n,
respectively; the cleavage site of the type IIs restriction
endonuclease recognition site of item (iii) of entry DNA n is
complementary to the cleavage site of the type IIs restriction
endonuclease recognition site of item (i) of entry DNA 1; said
system further comprising a destination vector comprising in this
order: (I) a type IIs restriction endonuclease recognition site
followed by the cleavage site thereof; (II) a vector backbone
comprising a selectable marker gene, said vector backbone linking
the cleavage sites of said recognition sites of items (I) and the
following item (III); (III) a further cleavage site of a type IIs
restriction endonuclease recognition site followed by the
recognition site of said cleavage site, and (IV) optionally, an
insert between the recognition sites of item (III) and item
(I).
2. The system according to claim 1, wherein a type IIs restriction
endonuclease recognising the recognition site (I) of said
destination vector can produce a single-stranded overhang from the
cleavage site of item (I) that is complementary to the
single-stranded overhang producible by the type IIs restriction
endonuclease recognising the recognition site (i) of entry DNA
numbered 1 for enabling annealing of said complementary
single-stranded overhangs and ligation of said destination vector
with the DNA segment of item (ii) from entry DNA numbered 1.
3. The system according to claim 1, said system comprising a
nucleic acid linker comprising in the following order: (a) a type
IIs restriction endonuclease recognition site; (b) a cleavage site
of said recognition site of item (a); (c) a cleavage site of a
further type IIs restriction endonuclease recognition site of the
following item (d); (d) a type IIs restriction endonuclease
recognition site defining the cleavage site of item (c) and being a
recognition site of a type IIs restriction endonuclease different
from that of item (a); (e) a type IIs restriction endonuclease
recognition site, preferably of the same endonuclease as the
recognition site of item (d); (f) a cleavage site of said
recognition site of item (e); (g) a cleavage site of a further type
IIs restriction endonuclease recognition site of the following item
(h); (h) a type IIs restriction endonuclease recognition site
defining the cleavage site of item (g), preferably of the same
endonuclease as the recognition site of item (a); said linker being
capable of linking a cleavage site of item (iii) of one of a entry
DNA numbered 1 to n, preferably of number 1 to n-1, to a cleavage
site of item (III) of said destination vector.
4. The system according to claim 3, wherein the cleavage site of
item (iii) of one of said entry DNAs is complementary to the
cleavage site of item (b) of said linker, and the cleavage site of
item (g) of said linker is complementary to the cleavage site of
item (III) of said destination vector.
5. The system according to claim 1, comprising from 1 to n multiple
destination vectors numbered 1 to n, each of said 1 to n
destination vectors having segments (I) to (III) as defined in
claim 1 and optionally a segment (IV) as defined in claim 1,
wherein the cleavage sites of item (III) of all n destination
vectors are identical and all cleavage sites of item (I) of all n
destination vectors are unique among the cleavage sites of item
(I).
6. The system according to claim 3, comprising a set of n nucleic
acid linkers numbered 1 to n, each n-th linker comprising items (a)
to (h) as defined in claim 3, the cleavage site of item (iii) of
each n-th entry DNA is complementary to the cleavage site of item
(b) of the n-th linker; the cleavage site of item (g) of each n-th
linker being complementary to the cleavage site of item (III) of
the n-th destination vector; whereby each n-th linker being capable
of linking a cleavage site of item (iii) of the n-th entry DNA to a
cleavage site of item (III) of each n-th destination vector.
7. The system according to claim 1, wherein each sequence portion
of item (ii) of each entry DNA 1 to n comprises a further pair of
two type IIs restriction endonuclease recognition sites oriented
such that said further pair of recognition sites can be removed
from said entry DNAs by treatment with type IIs restriction
endonuclease(s) recognising said further pair of recognition sites,
said further pair of recognition sites may flank a marker gene for
enabling selection of cell clones for the presence or absence of
said marker gene; wherein said further pair of two type IIs
restriction endonuclease recognition sites are recognition sites of
endonucleases different from the recognition sites of item (i) and
item (iii) of claim 1.
8. The system according to claim 1, wherein the cleavage sites of
the recognition sites of item (i) are unique among the item (i)
recognition sites of the set of n entry DNAs, and the cleavage
sites of the recognition sites of item (iii) are unique among the
item (iii) recognition sites within the set of n entry DNAs.
9. The system according to claim 1, wherein the type IIs
restriction endonuclease recognition sites of items (i) and (iii)
are recognition sites of the same type IIs restriction
endonuclease.
10. The system according to claim 1, wherein the cleavage sites of
the recognition sites of item (III) of all destination vectors are
identical, and the cleavage sites of the recognition sites of item
(I) of all destination vectors are non-identical.
11. A method of producing a nucleic acid construct of interest from
at least m nucleic acid fragment constructs numbered 1 to m, m
being an integer of at least 3; said method comprising the
following steps (A) to (C): (A) providing said m nucleic acid
fragment constructs, each of said m nucleic acid fragment
constructs comprising in this order: (i') a type IIs restriction
endonuclease recognition site of the upstream cleavage site of item
(ii'); (ii') a sequence segment of said nucleic acid construct of
interest, said sequence segment comprising, in this order, an
upstream cleavage site of the recognition site of item (i'), a core
portion of the sequence segment, and a downstream cleavage site of
the recognition site of the following item (iii'), and (iii') a
type IIs restriction endonuclease recognition site of said
downstream cleavage site of item (ii'); the downstream cleavage
sites of nucleic acid fragment constructs 1 to m-1 are
complementary to the upstream cleavage sites of nucleic acid
fragment constructs 2 to m, respectively, the downstream cleavage
site of a nucleic acid fragment construct u, wherein u is an
integer that is <m and at least 2, is complementary to the
upstream cleavage site of the type IIs restriction endonuclease
recognition site of item (ii') of nucleic acid fragment 1; (B)
reacting nucleic acid fragment constructs 1 to s, wherein s is an
integer <u, a destination vector and a linker in the presence of
a type IIs restriction endonuclease recognising said type IIs
restriction endonuclease recognition sites of items (i') and (iii')
and items (I) and (III) of the destination vector defined below and
in the presence a DNA ligase in reaction medium compatible with
activity of said type IIs restriction endonuclease and said ligase
for recombining and ligating, in the following order, the sequence
segment(s) of item (ii') of nucleic acid fragment constructs 1 to s
and said linker into said destination vector; said destination
vector comprising in this order: (I) a type IIs restriction
endonuclease recognition site followed by the cleavage site thereof
complementary to the upstream cleavage site of item (ii') of
nucleic acid fragment construct 1; (II) a vector backbone
comprising a selectable marker gene, said vector backbone linking
the cleavage sites of said recognition sites of items (I) and the
following item (III); (III) the further cleavage site of a type IIs
restriction endonuclease recognition site followed by the
recognition site of said cleavage site, said linker being as
defined in claim 3, wherein cleavage site (b) of said linker is
complementary to the downstream cleavage site of item (ii') of
nucleic acid fragment construct s, and wherein cleavage site (g) of
said linker and the cleavage site of item (III) of the destination
vector are complementary; and (C) treating a mixture comprising the
recombination product of step (B) and nucleic acid fragment
construct(s) s+1 to m with a type IIs restriction endonuclease
recognising said type IIs restriction endonuclease recognition
sites of items (i') and (iii'), a type IIs restriction endonuclease
recognising said type IIs restriction endonuclease recognition
sites of items (d) and (e) of the linker and a DNA ligase in a
reaction medium compatible with activity of said type IIs
restriction endonucleases and said ligase for inserting the
sequence segments of item (ii') of nucleic acid fragment constructs
s+1 to m and optionally a further linker as defined in claim 3 into
the cleavage sites provided by items (c) and (f) of the linker used
in step (B).
12. The method according to claim 11, wherein the recognition sites
of all nucleic acid fragment constructs of items (i') and (iii'),
the recognition sites of items (a) and (h) of the linker and the
recognition sites of item (I) and (III) of the destination vector
are recognition sites of the same type IIs restriction
endonuclease.
13. System for producing a nucleic acid construct of interest, said
system comprising: a set of n entry DNAs numbered 1 to n, n being
an integer of at least 3, each of said n entry DNAs comprising in
this order: (i) a type IIs restriction endonuclease recognition
site followed by the cleavage site thereof; (ii) a sequence portion
linking the cleavage site of said recognition site of item (i) with
the cleavage site of the recognition site of the following item
(iii), and (iii) a cleavage site of a further type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site; the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (iii) of entry DNAs 1 to n-1
are complementary to the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (i) of entry DNAs 2 to n,
respectively; all cleavages sites of item (i) are unique among said
n entry DNAs, and all cleavage sites of item (iii) are unique among
said n entry DNAs; said system further comprising a destination
vector comprising in this order: (I) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; (II) a vector backbone comprising a selectable marker
gene, said vector backbone linking the cleavage sites of said
recognition sites of items (I) and the following item (III); (III)
a further cleavage site of a type IIs restriction endonuclease
recognition site followed by the recognition site of said cleavage
site, and (IV) optionally, a linker between the recognition sites
of item (III) and item (I); said system further comprising a
nucleic acid linker comprising in the following order: (a) a type
IIs restriction endonuclease recognition site; (b) a cleavage site
of said recognition site of item (a); (c) a cleavage site of a
further type IIs restriction endonuclease recognition site of the
following item (d); (d) a type IIs restriction endonuclease
recognition site defining the cleavage site of item (c) and being a
recognition site of a type IIs restriction endonuclease different
from that of item (a); (e) a type IIs restriction endonuclease
recognition site, preferably of the same endonuclease as the
recognition site of item (d); (f) a cleavage site of said
recognition site of item (e); (g) a cleavage site of a further type
IIs restriction endonuclease recognition site of the following item
(h); (h) a type IIs restriction endonuclease recognition site
defining the cleavage site of item (g), preferably of the same
endonuclease as the recognition site of item (a); said linker being
capable of linking a cleavage site of item (iii) of one of a entry
DNA numbered 1 to n, preferably of number 1 to n-1, to a cleavage
site of item (III) of said destination vector.
14. The system according to claim 13, comprising the same number n
of said linkers as the system comprises entry DNAs, said linkers
being numbered 1 to n, wherein all linkers have the same cleavage
site (g) that is complementary to the cleavage site of item (III)
of said destination vector for linking each linker to the
recognition site of item (III) of said destination vector, and
wherein each of said n linkers has a different cleavage site (b)
that is complementary to the cleavage site of item (iii) of one of
said n entry DNAs.
15. The system according to claim 14, said system further
comprising n different destination vectors, each destination vector
being defined by items (I) to (IV) and having the same cleavage
site of item (III) that is complementary to the cleavage site (g)
of all linkers, each of said destination vectors having a different
cleavage site if item (I) that is complementary to the cleavage
site of item (i) of one of said n entry DNAs.
16. System for producing a nucleic acid construct of interest, said
system comprising: a set of n destination vectors ("destination
vectors M"), n being an integer of at least 2, preferably at least
3, each of said n destination vectors M comprising in the following
order: (I') a type IIs restriction endonuclease recognition site
defining the cleavage site of item (II'); (II') the cleavage site
of said recognition site of item (I'); (III') a cleavage site of
said recognition site of the following item (IV'); (IV') a further
type IIs restriction endonuclease recognition site defining the
cleavage site of item (III') and being a different recognition site
of a type IIs restriction endonuclease from that of item (I'); (V')
a vector backbone comprising a selectable marker gene, said vector
backbone linking the cleavage sites of said recognition sites of
item and (IV') and the following item (VI'); (VI') a further type
IIs restriction endonuclease cleavage site; (VII') a type IIs
restriction endonuclease recognition site of the cleavage site of
item (VI') and (VIII') optionally, an insert between the
recognition sites of item (VII') and item (I'); and a set of n
linkers M, n being as defined above, each linker M comprising in
the following order: (a') a type IIs restriction endonuclease
recognition site defining the cleavage site of item (b'); (b') the
cleavage site of said recognition site of item (a'); (c') a
cleavage site of a further type IIs restriction endonuclease
recognition site of item (d'), said cleavage site having the same
sequence of nucleotides as the cleavage site of item (b'); (d') the
type IIs restriction endonuclease recognition site defining the
cleavage site of item (c') and being a different recognition site
of a type IIs restriction endonuclease different from that of item
(a'); (e') a further cleavage site of a type IIs restriction
endonuclease recognition site of the following item (f'); (f') the
type IIs restriction endonuclease recognition site defining the
cleavage site of item (e'), that is preferably a recognition site
of the same endonuclease as the recognition site of item (a');
wherein the cleavage sites (VI') of all n destination vectors M are
identical; the cleavage sites (e') of all n linkers M are
identical; the cleavage site of item (VI') of each destination
vector M is complementary to the cleavage site of item (e') of each
linker M for allowing annealing of single-stranded overhangs
produced by the type IIs restriction endonuclease recognising
recognition sites (VII') and (f'); the cleavage sites of items
(II') and (III') within each destination vector M have the same
sequence of nucleotides and may overlap such that one and the same
sequence of nucleotides provides the cleavage site of items (II')
and that of item (III'); and the cleavage sites of items (b') and
(c') within each linker M have the same sequence of nucleotides and
may overlap such that one and the same sequence of nucleotides
provides the cleavage site of items (b') and that of item (c'); and
the cleavage site (II') of each destination vector M is unique
among the cleavage sites (II') of the set of n destination vectors
M such that there are n different cleavage sites (II'), wherein for
each of said n different cleavage sites (II'), there is a linker M
having a cleavage site (b') of identical nucleotide sequence among
the set of n linkers M.
17. The system according to claim 16, wherein (.alpha.) the
recognition sites of items (a') and (f') of all n linkers M are
recognition sites of the same type IIs restriction endonuclease;
(.beta.) the recognition sites of items (d') of all n linkers M are
recognition sites of the same type IIs restriction endonuclease;
wherein the recognition sites of item (.alpha.) are different
recognitions sites from those of item (.beta.).
18. The system according to claim 16, wherein (.gamma.) the
recognition sites of items (I') and (VII') of all n destination
vectors M are recognition sites of the same type IIs restriction
endonuclease; (.delta.) the recognition sites of items (IV') of all
n destination vectors M are recognition sites of the same type IIs
restriction endonuclease; wherein the recognition sites of item
(.gamma.) are different recognitions sites from those of item
(.delta.).
19. The system according to claim 16, wherein the recognition sites
of items (VII') and (I') of destination vectors M and of items (a')
and (f') of the linkers M are recognition sites of the same type
IIs restriction endonuclease; the recognition sites of items (IV')
of destination vectors M and of items (d') of the linkers M are
recognition sites of the same type IIs restriction
endonuclease.
20. The system according to claim 16, comprising: a set of z entry
DNAs numbered 1 to z, z being an integer of at least 2, preferably
an integer of at least 3, each of said z entry DNAs comprising in
this order: (i) a type IIs restriction endonuclease recognition
site followed by the cleavage site thereof; (ii) a sequence portion
linking the cleavage site of said recognition site of item (i) with
the cleavage site of the recognition site of the following item
(iii), and (iii) a cleavage site of a further type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site; wherein the cleavage site of item (i) of each
entry DNA is complementary to the cleavage site of item (II') of
one of the n destination vectors M for allowing annealing of
single-stranded overhangs produced by the type IIs restriction
endonuclease recognising recognition sites of items (i) and (I'),
the recognition sites of item (i) of all z entry DNAs are
preferably recognition sites of the same type IIs restriction
endonuclease as the recognition sites of item (I') and (VII'); the
cleavage site of item (iii) of each entry DNA is complementary to
the cleavage sites of item (b') of one of the n linkers M for
allowing annealing of single-stranded overhangs produced by the
type IIs restriction endonuclease recognising recognition sites of
items (iii) and (a'), the recognition sites of item (i) are
recognition sites of the same type IIs restriction endonuclease as
the recognition sites of item (a') and (f'); and the recognition
sites of items (i) and (iii) of all z entry DNAs are recognition
sites of the same type IIs restriction endonuclease.
21. The system according to claim 16, further comprising a set of n
destination vectors ("destination vectors P"), wherein n is as
defined in claim 1, each of said n destination vectors P comprising
in the following order: (I'') a type IIs restriction endonuclease
recognition site defining the cleavage site of item (II''); (II'')
the cleavage site of said recognition site of item (I''); (III'') a
cleavage site of said recognition site of the following item
(IV''); (IV'') a further type IIs restriction endonuclease
recognition site defining the cleavage site of item (III'') and
being a different recognition site of a type IIs restriction
endonuclease from that of item (I''); (V'') a vector backbone
comprising a selectable marker gene, said vector backbone linking
the cleavage sites of said recognition sites of item and (IV'') and
the following item (VI''); (VI'') a further type IIs restriction
endonuclease cleavage site; (VII'') a type IIs restriction
endonuclease recognition site of the cleavage site of item (VI''),
preferably of the same endonuclease as the recognition site of item
(I'') and (VIII'') optionally, an insert between the recognition
sites of item (VII'') and item (I''); and a set of n linkers P,
each linker P comprising in the following order: (a'') a type IIs
restriction endonuclease recognition site defining the cleavage
site of item (b''); (b'') the cleavage site of said recognition
site of item (a''); (c'') a cleavage site of a further type IIs
restriction endonuclease recognition site of item (d''), said
cleavage site having the same nucleotide sequence as the cleavage
site of item (b''); (d'') the type IIs restriction endonuclease
recognition site defining the cleavage site of item (c'') and being
a different recognition site of a type IIs restriction endonuclease
from that of item (a''); (e'') a further cleavage site of a type
IIs restriction endonuclease recognition site of the following item
(f''); (f'') the type IIs restriction endonuclease recognition site
defining the cleavage site of item (e''), that is preferably a
recognition site of the same endonuclease as the recognition site
of item (a''); wherein the cleavage sites (VI'') of all n
destination vectors P are identical; the cleavage sites (e'') of
all n linkers P are identical; the cleavage site of item (VI'') of
each destination vector P is complementary to the cleavage site of
item (e'') of each linker P for allowing annealing of
single-stranded overhangs produced by the type IIs restriction
endonuclease recognising recognition sites (VII'') and (f''); the
cleavage sites of items (II'') and (III'') within each destination
vector P have the same sequence of nucleotides and may overlap such
that one and the same sequence of nucleotides provides the cleavage
site of item (II'') and the cleavage site of item (III''); the
cleavage sites of items (b'') and (c'') within each linker P have
the same sequence of nucleotides and may overlap such that one and
the same sequence of nucleotides provides the cleavage site of
items (b'') and the cleavage site of item (c''); and for each of
said n different cleavage sites (b') or (II'), there is a
destination vector P having a cleavage site (II'') of identical
nucleotide sequence as the nucleotide sequence of cleavage sites
(b') or (II'); and for each of said n different cleavage sites (b')
or (II'), there is a linker P having a cleavage site (b'') of
identical nucleotide sequence as the nucleotide sequence of
cleavage sites (b') or (II').
22. The system according to claim 21, wherein the recognition sites
of items (I'') and (VII'') of all n destination vectors P are
recognition sites of the same type IIs restriction endonuclease;
and the recognition sites of items (IV'') of all n destination
vectors P are recognition sites of the same type IIs restriction
endonuclease but different from the recognition sites of items
(I'') and (VII'').
23. The system according to claim 21, wherein the recognition sites
of items (I''), (IV'), (d'), (a'') and (f'') are recognition sites
of the same type IIs restriction endonuclease; the recognition
sites of items (IV''), (I'), (VII'), (a') and (f') are recognition
sites of the same type IIs restriction endonuclease.
24. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a cloning system for
producing a nucleic acid construct of interest using type IIs
restriction endonucleases. The invention also provides a method of
producing a nucleic acid construct of interest from at least three
nucleic acid fragment constructs.
BACKGROUND OF THE INVENTION
[0002] Synthetic biology promises to revolutionize biotechnology
through engineering of life forms with novel phenotypes not
normally found in nature. Examples of applications include the
microbial production of chemical precursors, novel antibiotics and,
induction and fine-tuning of pluripotent stem cells and the
engineering of a minimal free living cell. Such applications will
require the ability to physically assemble complex DNA molecules
containing large numbers of natural or artificial genes in a wide
variety of arrangements.
[0003] Although much progress has been made in the past few years,
construction of recombinant DNA molecules is still a slow and
labour-intensive process. Recombinant DNA molecules have
traditionally been constructed using type II restriction enzymes
and ligase. Although versatile, such approach is slow and tedious
and only allows creation of constructs of relatively small size and
containing only few genes. In particular, this approach is limited
by the fact that designing cloning strategies becomes extremely
difficult for large constructs, since all restriction enzymes
available will cut many times in such constructs. In the past few
years, a number of different approaches have been developed to
overcome these limitations. These include recombinase-based
cloning, ligation-independent cloning, cloning based of homologous
recombination and PCR-based assembly. Recombinase-based cloning
eliminates the problems coming from the multiple occurrence of
restriction sites in large constructs but is limited by the fact
that recombination sites are left in the final construct,
preventing the seamless assembly of protein coding sequences.
Moreover, recombinase-based cloning is limited by the fact that, so
far, only 4 fragments can be assembled in one construct
simultaneously. Ligation-independent cloning is also independent of
restriction sites since restriction enzymes are not used, but is
limited by the fact that it requires PCR and therefore requires
sequencing of constructs made with this approach. Methods based on
homologous recombination are valuable and allow to assemble
extremely large DNA fragments of up to the size of entire bacterial
genomes (Gibson et al., Science. 2010 Jul. 2; 329(5987):52-6), but
are not well suited for combinatorial assembly of multiple
independent basic genetic elements since they, but are not well
suited for combinatorial assembly of multiple independent basic
genetic elements, since they require a minimum amount of sequence
in common between modules.
[0004] Recently, cloning methods based on type IIs restriction
enzymes have been developed (WO 2008/095927). Engler et al. PLoS
ONE 4 (2009) e5553) describe a protocol to assemble in one step and
one tube at least nine separate DNA fragments together into an
acceptor vector using type IIs restriction enzymes by simply
subjecting a mix of 10 undigested input plasmids to a
restriction-ligation reaction and transforming the resulting mix
into competent cells. This protocol was named "Golden Gate"
cloning.
[0005] Although methods that allow assembly of multiple DNA
fragments in one step (such as Golden Gate cloning) are helpful for
construction of recombinant DNA molecules, they still do not solve
the problem that construct-specific cloning strategies need to be
defined at each step of cloning. What is needed for synthetic
biology are methods that eliminate the need for construct-specific
cloning strategies. A step toward standardization of cloning
strategies has been proposed with the BioBrick system (Knight TF,
2003, Idempotent Vector Design for Standard Assembly of BioBricks.
MIT Synthetic Biology Working Group Technical Report). This system
is based on hierarchical assembly of basic genetic parts, two parts
at a time. Assembly of two basic parts using restriction enzymes
and ligase results in a composite part that has the same structure
as the basic part in terms of flanking restriction sites (the parts
are therefore called idempotent). Since the structure of the
composite parts is the same as the structure of the basic parts,
the same assembly procedure can be repeated on composite parts to
get increasingly complex constructs. However, because this strategy
is based on idempotency of the DNA fragments, the BioBrick system
is necessarily limited to assembly of two fragments at a time
(addition of a part to a plasmid that already contains another part
or composite part). This is a serious limitation since synthetic
biology will require assembling very large number of DNA fragments,
which will be very costly and impractical if assembly is performed
two fragments at a time. Moreover, the ability to assemble a large
number of fragments in a single step is useful for making
combinatorial libraries, for example for making a construct
containing all the genes encoding for a biochemical pathway. Such
library can be made by assembling in one step all the genes
necessary for a biochemical pathway, with multiple variants for
each of the genes of the pathway.
GENERAL DESCRIPTION OF THE INVENTION
[0006] Departing from the prior art, it is an object of this
invention to provide a system that overcomes the limitations of the
prior art. Notably, it is an object to provide a system and method
that is not limited to the combination of two fragments per
reaction. It is another object to provide a system of DNA molecules
that allows assembly of a large or even unlimited number of DNA
fragments using a fixed set of cloning vectors. The system should
allow assembly of multiple DNA fragments at each cloning step, and
should allow as many successive steps of cloning as necessary to be
performed, continually reusing the same set of vectors. Repetition
of these cloning cycles should allow assembly of increasingly
larger numbers of DNA fragments in any desired order, resulting in
increasingly larger constructs.
[0007] Accordingly, the present invention provides: [0008] (1)
System for producing a nucleic acid construct of interest, said
system comprising: [0009] a set of n entry DNAs numbered 1 to n, n
being an integer of at least 2, each of said n entry DNAs
comprising in this order: [0010] (i) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; [0011] (ii) a sequence portion linking the cleavage site
of said recognition site of item (i) with the cleavage site of the
recognition site of the following item (iii), and [0012] (iii) a
cleavage site of a further type IIs restriction endonuclease
recognition site followed by the recognition site of said cleavage
site; [0013] the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (iii) of entry DNAs 1 to n-1
are complementary to the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (i) of entry DNAs 2 to n,
respectively; [0014] the cleavage site of the type IIs restriction
endonuclease recognition site of item (iii) of entry DNA n is
complementary to the cleavage site of the type IIs restriction
endonuclease recognition site of item (i) of entry DNA 1 for
allowing annealing of complementary single-stranded overhangs
formed by restriction at recognition site (i) of entry DNA 1 and at
recognition site (iii) of entry DNA n; [0015] said system further
comprising a destination vector comprising in this order: [0016]
(I) a type IIs restriction endonuclease recognition site followed
by the cleavage site thereof; [0017] (II) a vector backbone
preferably comprising a selectable marker gene, said vector
backbone linking the cleavage sites of said recognition sites of
items (I) and the following item (III); [0018] (III) a further
cleavage site of a type IIs restriction endonuclease recognition
site followed by the recognition site of said cleavage site, and
[0019] (IV) optionally, an insert between the recognition sites of
item (III) and item (I); [0020] said cleavage sites of items (I)
and (III) being different and non-complementary, said recognition
sites of items (I) and (III) being preferably recognitions sites of
the same endonuclease. [0021] (2) The system according to (1),
wherein a type IIs restriction endonuclease recognising the
recognition site (I) of said destination vector can produce a
single-stranded overhang from the cleavage site of item (I) that is
complementary to the single-stranded overhang producible by the
type IIs restriction endonuclease recognising the recognition site
(i) of entry DNA numbered 1 for enabling annealing of said
complementary single-stranded overhangs and ligation of said
destination vector with the DNA segment of item (ii) from entry DNA
numbered 1. [0022] (3) The system according to (1) or (2), said
system comprising a nucleic acid linker comprising in the following
order: [0023] (a) a type IIs restriction endonuclease recognition
site; [0024] (b) a cleavage site of said recognition site of item
(a); [0025] (c) a cleavage site of a further type IIs restriction
endonuclease recognition site of the following item (d); [0026] (d)
a type IIs restriction endonuclease recognition site defining the
cleavage site of item (c) and being a recognition site of a type
IIs restriction endonuclease different from that of item (a);
[0027] (e) a type IIs restriction endonuclease recognition site,
preferably of the same endonuclease as the recognition site of item
(d); [0028] (f) a cleavage site of said recognition site of item
(e); [0029] (g) a cleavage site of a further type IIs restriction
endonuclease recognition site of the following item (h); [0030] (h)
a type IIs restriction endonuclease recognition site defining the
cleavage site of item (g), preferably of the same endonuclease as
the recognition site of item (a); [0031] said linker being capable
of linking a cleavage site of item (iii) of one of a entry DNA
numbered 1 to n, preferably of number 1 to n-1, to a cleavage site
of item (III) of said destination vector. [0032] (4) The system
according to (3), wherein the cleavage site of item (iii) of one of
said entry DNAs is complementary to the cleavage site of item (b)
of said linker, and [0033] the cleavage site of item (g) of said
linker is complementary to the cleavage site of item (III) of said
destination vector. [0034] (5) The system according to any one of
(1) to (4), comprising from 1 to n multiple destination vectors
numbered 1 to n, each of said 1 to n destination vectors having
segments (I) to (III) as defined in claim 1 and optionally a
segment (IV) as defined in claim 1, [0035] wherein the cleavage
sites of item (III) of all destination vectors are identical and
all cleavage sites of item (I) of all destination vectors are
unique among the cleavage sites of item (III). [0036] (6) The
system according to (3), comprising a set of n nucleic acid linkers
numbered 1 to n, each n-th linker comprising items (a) to (h) as
defined in claim 3, [0037] the cleavage site of item (iii) of each
n-th entry DNA is complementary to the cleavage site of item (b) of
the n-th linker; [0038] the cleavage site of item (g) of each n-th
linker being complementary to the cleavage site of item (III) of
the n-th destination vector; [0039] whereby each n-th linker being
capable of linking a cleavage site of item (iii) of the n-th entry
DNA to a cleavage site of item (III) of each n-th destination
vector. [0040] (7) The system according to any one of (3), (4) or
(6), said linker(s) comprising a marker gene in between items (d)
and (e) for enabling selection of cell clones for the presence or
absence of said marker gene. [0041] (8) The system according to any
one of (1) to (7), wherein each sequence portion of item (ii) of
each entry DNA 1 to n comprises a further pair of two type IIs
restriction endonuclease recognition sites oriented such that said
further pair of recognition sites can be removed from said entry
DNAs by treatment with type IIs restriction endonuclease(s)
recognising said further pair of recognition sites, said further
pair of recognition sites may flank a marker gene for enabling
selection of cell clones for the presence or absence of said marker
gene; [0042] wherein said further pair of two type IIs restriction
endonuclease recognition sites are recognition sites of
endonucleases different from the recognition sites of item (i) and
item (iii) of claim 1. [0043] (9) The system according to any one
of (1) to (8), wherein the cleavage sites of the recognition sites
of item (i) are unique among the item (i) recognition sites of the
set of n entry DNAs, and the cleavage sites of the recognition
sites of item (iii) are unique among the item (iii) recognition
sites within the set of n entry DNAs. [0044] (10) The system
according to any one of (1) to (9), each of said n entry DNAs
further comprising in the order defined in claim 1: [0045] (iv) a
vector backbone comprising a selectable marker gene. [0046] (11)
The system according to any one of (1) to (10), wherein the type
IIs restriction endonuclease recognition sites of items (i) and
(iii) are recognition sites of the same type IIs restriction
endonuclease. [0047] (12) The system according to any one of (1) to
(11), wherein the cleavage sites of the recognition sites of item
(III) of all destination vectors are identical, and the cleavage
sites of the recognition sites of item (I) of all destination
vectors are non-identical. [0048] (13) The system according to (3),
wherein the cleavage sites of items (b) and (c) have the same
sequence and preferably overlap, and wherein the cleavage sites of
items (f) and (g) have the same sequence and preferably overlap.
[0049] (14) A method of producing a nucleic acid construct of
interest from at least m nucleic acid fragment constructs numbered
1 to m, each nucleic acid construct of interest comprising a
sequence segment numbered 1 to m in the order of occurrence in the
nucleic acid construct of interest, m being an integer of at least
3; [0050] said method comprising the following steps (A) to (C):
[0051] (A) providing said m nucleic acid fragment constructs, each
of said m nucleic acid fragment constructs comprising in this
order: [0052] (i') a type IIs restriction endonuclease recognition
site of the upstream cleavage site of item (ii'); [0053] (ii') a
sequence segment of said nucleic acid construct of interest, said
sequence segment comprising, in this order, an upstream cleavage
site of the recognition site of item (i'), a core portion of the
sequence segment, and a downstream cleavage site of the recognition
site of the following item (iii'), and [0054] (iii') the type IIs
restriction endonuclease recognition site of said downstream
cleavage site of item (ii'); [0055] the downstream cleavage sites
of nucleic acid fragment constructs 1 to m-1 are complementary to
the upstream cleavage sites of nucleic acid fragment constructs 2
to m, respectively, [0056] the downstream cleavage site of a
nucleic acid fragment construct u, wherein u is an integer that is
<m and at least 2 is complementary to the upstream cleavage site
of the type IIs restriction endonuclease recognition site of item
(ii') of nucleic acid fragment 1; [0057] (B) combining nucleic acid
fragment constructs 1 to s, wherein s is an integer <u, a
destination vector and a linker in the presence of a type IIs
restriction endonuclease recognising said type IIs restriction
endonuclease recognition sites of items (i') and (iii') and items
(I) and (III) of the destination vector defined below and in the
presence a DNA ligase in reaction medium compatible with activity
of said type IIs restriction endonuclease and said ligase for
recombining and ligating, in the following order, the sequence
segment(s) of item (ii') of nucleic acid fragment constructs 1 to s
and said linker into said destination vector; [0058] said
destination vector comprising in this order: [0059] (I) a type IIs
restriction endonuclease recognition site followed by the cleavage
site thereof complementary to the upstream cleavage site of item
(ii') of nucleic acid fragment construct 1; [0060] (II) a vector
backbone comprising a selectable marker gene, said vector backbone
linking the cleavage sites of said recognition sites of items (I)
and the following item (III); [0061] (III) the further cleavage
site of a type IIs restriction endonuclease recognition site
followed by the recognition site of said cleavage site, [0062] said
linker being as defined in item (3), wherein cleavage site (b) of
said linker is complementary to the downstream cleavage site of
item (ii') of nucleic acid fragment construct s, and wherein
cleavage site (g) of said linker and the cleavage site of item
(III) of the destination vector are complementary; and [0063] (C)
treating a mixture comprising the recombination product of step (B)
and nucleic acid fragment construct(s) s+1 to m with a type IIs
restriction endonuclease recognising said type IIs restriction
endonuclease recognition sites of items (i') and (iii'), a type IIs
restriction endonuclease recognising said type IIs restriction
endonuclease recognition sites of items (d) and (e) of the linker
and a DNA ligase in a reaction medium compatible with activity of
said type IIs restriction endonucleases and said ligase for
inserting the sequence segments of item (ii') of nucleic acid
fragment constructs s+1 to m and optionally a further linker as
defined in item (3) into the cleavage sites provided by items (c)
and (f) of the linker used in step (B). [0064] (15) The method
according to (14), wherein the recognition sites of all nucleic
acid fragment constructs of items (i') and (iii'), the recognition
sites of items (a) and (h) of the linker and the recognition sites
of item (I) and (III) of the destination vector are recognition
sites of the same type IIs restriction endonuclease. [0065] (16)
System for producing a nucleic acid construct of interest, said
system comprising: [0066] a set of n entry DNAs numbered 1 to n, n
being an integer of at least 2, preferably at least 3, [0067] each
of said n entry DNAs comprising in this order: [0068] (i) a type
IIs restriction endonuclease recognition site followed by the
cleavage site thereof; [0069] (ii) a DNA segment linking the
cleavage site of said recognition site of item (i) with the
cleavage site of the recognition site of the following item (iii),
and [0070] (iii) a cleavage site of a further type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site; [0071] the cleavage sites of the type IIs
restriction endonuclease recognition sites of item (iii) of entry
DNAs 1 to n-1 are complementary to the cleavage sites of the type
IIs restriction endonuclease recognition sites of item (i) of entry
DNAs 2 to n, respectively; [0072] all cleavages sites of item (i)
are typically unique among said n entry DNAs, and all cleavage
sites of item (iii) are typically unique among said n entry DNAs;
[0073] said system further comprising a destination vector
comprising in this order: [0074] (I) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; [0075] (II) a vector backbone comprising a selectable
marker gene, said vector backbone linking the cleavage sites of
said recognition sites of items (I) and the following item (III);
[0076] (III) a further cleavage site of a type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site, and [0077] (IV) optionally, a linker between
the recognition sites of item (III) and item (I); said system
further comprising a nucleic acid linker comprising in the
following order: [0078] (a) a type IIs restriction endonuclease
recognition site; [0079] (b) a cleavage site of said recognition
site of item (a); [0080] (c) a cleavage site of a further type IIs
restriction endonuclease recognition site of the following item
(d); [0081] (d) a type IIs restriction endonuclease recognition
site defining the cleavage site of item (c) and being a recognition
site of a type IIs restriction endonuclease different from that of
item (a); [0082] (e) a type IIs restriction endonuclease
recognition site, preferably of the same endonuclease as the
recognition site of item (d); [0083] (f) a cleavage site of said
recognition site of item (e); [0084] (g) a cleavage site of a
further type IIs restriction endonuclease recognition site of the
following item (h); [0085] (h) a type IIs restriction endonuclease
recognition site defining the cleavage site of item (g), preferably
of the same endonuclease as the recognition site of item (a);
[0086] said linker being capable of linking a cleavage site of item
(iii) of one of a entry DNA numbered 1 to n, preferably of number 1
to n-1, to a cleavage site of item (III) of said destination
vector. [0087] (17) The system according to (16), comprising the
same number n of said linkers as the system comprises entry DNAs,
said linkers being numbered 1 to n,
[0088] wherein all linkers have the same cleavage site (g) that is
complementary to the cleavage site of item (III) of said
destination vector for linking each linker to the recognition site
of item (III) of said destination vector, and [0089] wherein each
of said n linkers has a different cleavage site (b) that is
complementary to the cleavage site of item (iii) of one of said n
entry DNAs. [0090] (18) The system according to (17), said system
further comprising n different destination vectors, each
destination vector being defined by items (I) to (IV) and having
the same cleavage site of item (III) that is complementary to the
cleavage site (g) of all linkers, [0091] each of said destination
vectors having a different cleavage site if item (I) that is
complementary to the cleavage site of item (i) of one of said n
entry DNAs. [0092] (19) System for producing a nucleic acid
construct of interest, said system comprising: [0093] a set of m
nucleic acid fragment constructs numbered 1 to m, m being an
integer of at least 2, preferably at least 3, [0094] each of said m
nucleic acid fragment constructs comprising in this order: [0095]
(i') a type IIs restriction endonuclease recognition site of the
upstream cleavage site of item (ii'); [0096] (ii') a sequence
segment of said nucleic acid construct of interest, said sequence
segment comprising, in this order, an upstream cleavage site of the
recognition site of item (i'), a core portion of the sequence
segment, and a downstream cleavage site of the recognition site of
the following item (iii'), and [0097] (iii') a type IIs restriction
endonuclease recognition site of said downstream cleavage site of
item (ii'); [0098] the downstream cleavage sites of nucleic acid
fragment constructs 1 to m-1 are complementary to the upstream
cleavage sites of nucleic acid fragment constructs 2 to m,
respectively; [0099] said system further comprising a destination
vector comprising in this order: [0100] (I) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; [0101] (II) a vector backbone comprising a selectable
marker gene, said vector backbone linking the cleavage sites of
said recognition sites of items (I) and the following item (III);
[0102] (III) a further cleavage site of a type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site, and [0103] (IV) optionally, an insert between
the recognition sites of item (III) and item (I); [0104] said
cleavage sites of items (I) and (III) being different and
non-complementary, said recognition sites of items (I) and (III)
being recognitions sites of the same endonuclease. [0105] (20) The
system of item (19), wherein the downstream cleavage site of a
nucleic acid fragment construct u, wherein u is an integer that is
<m and at least 2 is complementary to the upstream cleavage site
of the type IIs restriction endonuclease recognition site of item
(ii') of nucleic acid fragment 1. [0106] (21) System for producing
a nucleic acid construct of interest, said system comprising:
[0107] a set of n destination vectors ("destination vectors M"), n
being an integer of at least 2, preferably at least 3, [0108] each
of said n destination vectors M comprising in the following order:
[0109] (I') a type IIs restriction endonuclease recognition site
defining the cleavage site of item (II'); [0110] (II') the cleavage
site of said recognition site of item (I'); [0111] (III') a
cleavage site of said recognition site of the following item (IV');
[0112] (IV') a further type IIs restriction endonuclease
recognition site defining the cleavage site of item (III') and
being a different recognition site of a type IIs restriction
endonuclease from that of item (I'); [0113] (V') a vector backbone
comprising a selectable marker gene, said vector backbone linking
the cleavage sites of said recognition sites of item and (IV') and
the following item (VI'); [0114] (VI') a further type IIs
restriction endonuclease cleavage site; [0115] (VII') a type IIs
restriction endonuclease recognition site of the cleavage site of
item (VI'), preferably of the same type IIs restriction
endonuclease as the recognition site of item (I') and [0116]
(VIII') optionally, an insert between the recognition sites of item
(VII') and item (I'); and [0117] a set of n linkers M, n being as
defined above, each linker M comprising in the following order:
[0118] (a') a type IIs restriction endonuclease recognition site
defining the cleavage site of item (b'); [0119] (b') the cleavage
site of said recognition site of item (a'); [0120] (c') a cleavage
site of a further type IIs restriction endonuclease recognition
site of item (d'), said cleavage site having the same sequence of
nucleotides as the cleavage site of item (b'); [0121] (d') the type
IIs restriction endonuclease recognition site defining the cleavage
site of item (c') and being a different recognition site of a type
IIs restriction endonuclease different from that of item (a');
[0122] (e') a further cleavage site of a type IIs restriction
endonuclease recognition site of the following item (f'); [0123]
(f') the type IIs restriction endonuclease recognition site
defining the cleavage site of item (e'), that is preferably a
recognition site of the same endonuclease as the recognition site
of item (a'); [0124] wherein [0125] the cleavage sites (VI') of all
n destination vectors M are identical; [0126] the cleavage sites
(e') of all n linkers M are identical; the cleavage site of item
(VI') of each destination vector M is complementary to the cleavage
site of item (e') of each linker M for allowing annealing of
single-stranded overhangs produced by the type IIs restriction
endonuclease recognising recognition sites (VII') and (f'); [0127]
the cleavage sites of items (II') and (III') within each
destination vector M have the same sequence of nucleotides and may
overlap such that one and the same sequence of nucleotides provides
the cleavage site of items (II') and that of item (III'); and
[0128] the cleavage sites of items (b') and (c') within each linker
M have the same sequence of nucleotides and may overlap such that
one and the same sequence of nucleotides provides the cleavage site
of items (b') and that of item (c'); and [0129] the cleavage site
(II') of each destination vector M is unique among the cleavage
sites (II') of the set of n destination vectors M such that there
are n different cleavage sites (II'), wherein for each of said n
different cleavage sites (II'), there is a linker M having a
cleavage site (b') of identical nucleotide sequence among the set
of n linkers M. [0130] (22) The system according to item (21),
comprising: [0131] a set of z entry DNAs numbered 1 to z, z being
an integer of at least 2, preferably an integer of at least 3,
[0132] each of said z entry DNAs comprising in this order: [0133]
(i) a type IIs restriction endonuclease recognition site followed
by the cleavage site thereof; [0134] (ii) a sequence portion
linking the cleavage site of said recognition site of item (i) with
the cleavage site of the recognition site of the following item
(iii), and [0135] (iii) a cleavage site of a further type IIs
restriction endonuclease recognition site followed by the
recognition site of said cleavage site; [0136] wherein [0137] the
cleavage site of item (i) of each entry DNA is complementary to the
cleavage site of item (II') of one of the n destination vectors M
for allowing annealing of single-stranded overhangs produced by the
type IIs restriction endonuclease recognising recognition sites of
items (i) and (I'), [0138] the recognition sites of item (i) of all
z entry DNAs are preferably recognition sites of the same type IIs
restriction endonuclease as the recognition sites of item (I') and
(VII'); [0139] the cleavage site of item (iii) of each entry DNA is
complementary to the cleavage sites of item (b') of one of the n
linkers M for allowing annealing of single-stranded overhangs
produced by the type IIs restriction endonuclease recognising
recognition sites of items (iii) and (a'), [0140] the recognition
sites of item (i) are recognition sites of the same type IIs
restriction endonuclease as the recognition sites of item (a') and
(f'); and [0141] the recognition sites of items (i) and (iii) of
all z entry DNAs are recognition sites of the same type IIs
restriction endonuclease. [0142] (23) The system according to any
one of items (21) or (22), further comprising a set of n
destination vectors ("destination vectors P"), wherein n is as
defined in item (21), [0143] each of said n destination vectors P
comprising in the following order: [0144] (I'') a type IIs
restriction endonuclease recognition site defining the cleavage
site of item (II''); [0145] (II'') the cleavage site of said
recognition site of item (I''); [0146] (III'') a cleavage site of
said recognition site of the following item (IV''); [0147] (IV'') a
further type IIs restriction endonuclease recognition site defining
the cleavage site of item (III'') and being a different recognition
site of a type IIs restriction endonuclease from that of item
(I''); [0148] (V'') a vector backbone comprising a selectable
marker gene, said vector backbone linking the cleavage sites of
said recognition sites of item and (IV'') and the following item
(VI''); [0149] (VI'') a further type IIs restriction endonuclease
cleavage site; [0150] (VII'') a type IIs restriction endonuclease
recognition site of the cleavage site of item (VI''), preferably of
the same endonuclease as the recognition site of item (I'') and
[0151] (VIII'') optionally, an insert between the recognition sites
of item (VII'') and item (I''); and [0152] a set of n linkers P,
each linker P comprising in the following order: [0153] (a'') a
type IIs restriction endonuclease recognition site defining the
cleavage site of item (b''); [0154] (b'') the cleavage site of said
recognition site of item (a''); [0155] (c'') a cleavage site of a
further type IIs restriction endonuclease recognition site of item
(d''), said cleavage site having the same nucleotide sequence as
the cleavage site of item (b''); [0156] (d'') the type IIs
restriction endonuclease recognition site defining the cleavage
site of item (c'') and being a different recognition site of a type
IIs restriction endonuclease from that of item (a''); [0157] (e'')
a further cleavage site of a type IIs restriction endonuclease
recognition site of the following item (f''); (f'') the type IIs
restriction endonuclease recognition site defining the [0158]
cleavage site of item (e''), that is preferably a recognition site
of the same endonuclease as the recognition site of item (a'');
[0159] wherein [0160] the cleavage sites (VI'') of all n
destination vectors P are identical; [0161] the cleavage sites
(e'') of all n linkers P are identical; [0162] the cleavage site of
item (VI'') of each destination vector P is complementary to the
cleavage site of item (e'') of each linker P for allowing annealing
of single-stranded overhangs produced by the type IIs restriction
endonuclease recognising recognition sites (VII'') and (f'');
[0163] the cleavage sites of items (II'') and (III'') within each
destination vector P have the same sequence of nucleotides and may
overlap such that one and the same sequence of nucleotides provides
the cleavage site of item (II'') and the cleavage site of item
(III''); [0164] the cleavage sites of items (b'') and (c'') within
each linker P have the same sequence of nucleotides and may overlap
such that one and the same sequence of nucleotides provides the
cleavage site of items (b'') and the cleavage site of item (c'');
and [0165] for each of said n different cleavage sites (b') or
(II'), there is a destination vector P having a cleavage site
(II'') of identical nucleotide sequence as the nucleotide sequence
of cleavage sites (b') or (II'); and [0166] for each of said n
different cleavage sites (b') or (II'), there is a linker P having
a cleavage site (b'') of identical nucleotide sequence as the
nucleotide sequence of cleavage sites (b') or (II').
[0167] The system of the invention comprises a defined set of
components that have a high versatility and flexibility, whereby a
given system can be easily applied to many different applications.
Notably, a given system can be used for applications comprising
different numbers of fragments to be assembled in a nucleic acid
construct of interest. It is a great advantage of the invention
that many different fragments can be combined with a number of
acceptor vectors that is smaller than the number of fragments to be
combined. Therefore, the system can be scaled to the combination of
many different fragments and fragment numbers with little or no
extra cloning work for the adaption of acceptor vectors to a large
number of fragments.
[0168] This system provides three advantages: (1) the cloning
system allows to assemble constructs from multiple DNA fragments at
each cloning step (using Golden Gate cloning), (2) the cloning
procedure is automatically defined by the number of genetic
elements that the user wants to assemble and does not require
construct-specific cloning strategies and can therefore easily be
automatized, (3) the cloning procedure can be repeated indefinitely
using the same set of cloning vectors to make increasingly complex
constructs (with an increasingly higher number of multigene and/or
genetic elements.
[0169] In the invention, a nucleic acid construct of interest is a
DNA assembled from m nucleic acid fragment constructs, m being an
integer of at least 3. Each nucleic acid fragment construct
provides a sequence segment to the nucleic acid construct of
interest. Typically, the nucleic acid construct of interest is
present in a vector having in its backbone a selectable marker for
allowing selection of cells containing the vector. The nucleic acid
construct of interest is produced in the invention in a process
comprising at least two, typically three, steps of restriction and
ligation, for example departing from standardised pre-prepared
modules. Restriction is catalysed by a type IIs restriction
endonuclease, ligation is catalysed by a ligase.
[0170] In a first step of restriction and ligation corresponding to
step (A) of the method of the invention (also referred herein as
"level 1" or "level 1 reaction"), at least one, preferably at least
2, nucleic acid modules are linked by restriction and ligation and
at the same time inserted into an acceptor vector. Acceptor vectors
are referred to herein as "destination vectors". Thus, the acceptor
vectors of the level 1 reaction are also referred to herein as
"level 1 destination vectors" or "level 1 acceptor vector". The
level 1 acceptor or destination vectors are also referred to herein
as "entry DNAs". The reaction products of the level 1 reaction are
referred to as "level 1 constructs" or "nucleic acid fragment
constructs", the latter terms being equivalent herein. The term
"construct" herein indicates a reaction product of a restriction
and ligation reaction. Thus, a level 1 destination vector is a
reactant of the level 1 reaction, and the nucleic acid fragment
constructs are the products of the level 1 reaction. Multiple level
1 reactions are generally conducted separately to obtain at least
two different nucleic acid fragment constructs to be assembled in
the second step, referred to herein as "level 2" (see further
below). One purpose of the level 1 reaction is to provide the
nucleic acid fragment constructs to be assembled in the next step
with suitable cleavage sites of a type IIs restriction endonuclease
to allow ligation of the constructs obtained on level 1 in the
desired order on level 2. In some embodiments, the level 1 reaction
further serves the purpose of constructing nucleic acid fragment
constructs from 2 or more modules. For example, if the nucleic acid
construct of interest comprises several eukaryotic transcription
units, multiple individual transcription units can be assembled in
separate level 1 reactions from 2 or more modules (such as
promoter, 5' UTR, signal peptide sequence etc.). On level 2, two or
more transcription units can then be combined. In a further level 2
reaction, one or more further nucleic acid construct each
containing a transcription unit can be combined with the reaction
product of the first level 2 reaction. It is, however, not
compulsory to produce the nucleic acid fragment constructs using
such level 1 reaction. It could also be considered to engineer them
by other means or to synthesise them artificially de novo.
[0171] In the second step of restriction and ligation corresponding
to step (B) of the method of the invention (referred herein as
"level 2"), at least 2 nucleic acid fragment constructs obtained in
the previous level 1 step are combined by restriction and ligation
and, in the same reaction, inserted into an acceptor vector. This
acceptor vector of the level 2 reaction is referred to as "level 2
destination vector". If the term "destination vector" is used
without reference to a particular level, it refers to a level 2
destination vector. The reaction product of the level 2 reaction is
referred to as "level 2 construct". In some embodiments, the level
2 construct is the nucleic acid construct of interest. In other
embodiments, such as in the method of the invention, the level 2
reaction is followed by a further reaction step (step (C)) that may
be referred to as "level 2-2", indicating a second level 2
reaction. In the first level 2 reaction, a nucleic acid linker
(also simply referred to as "linker" or "end-linker" herein) is
preferably used that links one of the at least two nucleic acid
fragment constructs to one of the cleavage sites of the level 2
destination vector. Use of the linker or multiple linkers
significantly improves the versatility and flexibility of the
systems of the invention in that different nucleic acid fragment
constructs can be inserted into a given destination vector, whereby
a given destination vector can be used independent of the cleavage
site of the nucleic acid fragment construct. Moreover, the linkers
allow introduction of a type IIs restriction site for re-opening
the level 2 reaction product for insertion of further nucleic acid
fragment constructs in a further step of restriction and ligation
(step (C)) as will be described below.
[0172] The term "module" is used herein to refer to the starting
compound of a level 1 reaction other than the level 1 destination
vector. Thus, a module is a reactant of a restriction and ligation
reaction that reacts with a level 1 destination vector. The modules
of a level 1 reaction can be produced in a level 0 reaction (see
further below). Thus, the modules of the level 1 reaction can be
the products of a level 0 reaction.
[0173] The system of the invention comprises a set of n entry DNAs
and at least one destination vector. n is an integer of at least 2,
in another embodiment of at least 3. Conveniently, n may be between
3 and 10. In the figures, examples with sets of n=7 entry DNAs are
presented (FIG. 4). As mentioned above, the term "entry DNA" refers
to acceptor vectors of the level 1 reaction. The destination vector
is an acceptor vector of the level 2 reaction. Thus, the entry DNAs
and the at least one destination vector of the system are key
components for performing both the level 1 and the level 2
reaction. The entry DNAs of the system of the invention allow
combination of the multiple nucleic acid fragment constructs
produced from multiple entry DNAs in a desired order and insertion
into the level 2 destination vector in the level 2 reaction. The
entry DNAs are numbered consecutively with integers starting from 1
in the order in which inserts inserted into level 1 reaction can be
assembled into the nucleic acid construct of interest in a
subsequent step. Entry DNAs differing in their numbering by 1 are
referred to as contiguous entry DNAs. For allowing combination of
entry DNAs and/or fragment constructs derived from the entry DNAs
in a desired order in a level 2 reaction, each of said n entry DNAs
comprises in this order: [0174] (i) a type IIs restriction
endonuclease recognition site followed by the cleavage site
thereof; [0175] (ii) a sequence portion linking the cleavage site
of said recognition site of item (i) with the cleavage site of the
recognition site of the following item (iii), and [0176] (iii) a
cleavage site of a further type IIs restriction endonuclease
recognition site followed by the recognition site of said cleavage
site.
[0177] The cleavage sites of the type IIs restriction endonuclease
recognition sites of item (iii) of entry DNAs 1 to n-1 are
complementary to the cleavage sites of the type IIs restriction
endonuclease recognition sites of item (i) of entry DNAs 2 to n,
respectively. Being complementary means that single-stranded
overhangs produced by restriction with a type IIs restriction
endonuclease recognising the recognition sites of the cleavage
sites are complementary such that the single stranded overhangs can
anneal and be ligated after annealing to form a linear DNA. Thus,
the first entry DNA can anneal with its end represented by item
(iii) to the end represented by item (i) of the second entry DNA.
The second entry DNA can anneal with its end represented by item
(iii) to the end represented by item (i) of the third entry DNA
etc. This is illustrated by the dashed arrows in FIG. 4 that link
the right hand side of the level 1 destination vectors with the
left hand side of the level 1 destination vector underneath.
[0178] Generally, all item (i) cleavage sites are unique and
non-complementary among the n entry DNAs of the system of the
invention, and all item (iii) cleavage sites of all entry DNAs are
unique and non-complementary among the n entry DNAs of the
invention. In a given entry DNA, the cleavage sites of items (i)
and (iii) are preferably non complementary in order to avoid
ligating multiple identical fragment constructs contiguously. It is
also preferred that the recognition sites of items (i) and (iii)
among all entry DNAs are recognitions sites of the same
endonuclease so that the associated cleavage sites can be cleaved
using the same type IIs restriction endonuclease. However, it is
also possible that the recognition sites of different entry DNAs
are recognition sites of different type IIs restriction
endonucleases. In this case, multiple endonucleases will have to be
used in a given level 2 reaction to ensure that all required
cleavage sites are cleaved. The single-stranded overhangs formed
from the cleavage sites by type IIs restriction enzyme cleavage are
preferably non-palindromic.
[0179] In a preferred embodiment, the cleavage site of the type IIs
restriction endonuclease recognition site of item (iii) of entry
DNA n is complementary to the cleavage site of the type IIs
restriction endonuclease recognition site of item (i) of entry DNA
1 for allowing annealing of complementary single-stranded overhangs
formed by restriction at recognition site (i) of entry DNA 1 and at
recognition site (iii) of entry DNA n. This feature is illustrated
in the long dashed arrow linking the TGCC cleavage site of the
level 1 destination vector pL1F-7 with the TGCC cleavage site of
the level 1 destination vector pL1F-1 (FIG. 4). As will be
described in more detail below, this allows reuse of the first and
optionally further entry DNAs in a second level 2 reaction so that
more nucleic acid fragment constructs can be combined to produce a
nucleic acid construct of interest than the system has entry
DNAs.
[0180] The entry DNAs may be circular plasmids or vectors, wherein
items (i) and (iii) of the entry DNAs are linked by a vector
backbone. The vector backbone may contain a selectable marker
allowing selection of cell clones containing the entry DNA or the
nucleic acid fragment construct obtained therefrom in the level 1
reaction.
[0181] The system further comprises a destination vector (level 2
destination vector) comprising in this order: [0182] (I) a type IIs
restriction endonuclease recognition site followed by the cleavage
site thereof; [0183] (II) a vector backbone comprising a selectable
marker gene, said vector backbone linking the cleavage sites of
said recognition sites of items (I) and the following item (III);
[0184] (III) a further cleavage site of a type IIs restriction
endonuclease recognition site followed by the recognition site of
said cleavage site, and [0185] (IV) optionally, an insert between
the recognition sites of item (III) and item (I);
[0186] In the destination vector, the cleavage sites of items (I)
and (III) are different and non-complementary. Preferably, the
recognition sites of items (I) and (III) are recognition sites of
the same endonuclease so that the associated cleavage sites can be
cleaved using the same type IIs restriction endonuclease. For
convenience, the recognition sites of items (I) and (III) are
further recognitions sites of the same endonuclease as the
recognition sites of items (i) and (iii) of the entry DNAs, so that
the level 2 reaction can be performed using one type IIs
restriction endonuclease.
[0187] For enabling ligation of multiple nucleic acid fragment
constructs into the destination vector, the type IIs restriction
endonuclease recognising the recognition site (I) of said
destination vector can produce a single-stranded overhang from the
cleavage site of item (I) that is complementary to the
single-stranded overhang producible by the type IIs restriction
endonuclease recognising the recognition site (i) of entry DNA
numbered 1 for enabling annealing of said complementary
single-stranded overhangs and ligation of said destination vector
with the DNA segment of item (ii) from entry DNA numbered 1. In the
terminology used herein, the cleavage site of item (i) of entry DNA
1 and the cleavage site of item (I) of the destination vector are
complementary. Alternatively, the type IIs restriction endonuclease
recognising the recognition site (I) of a destination vector can
produce a single-stranded overhang from the cleavage site of item
(I) that is complementary to the single-stranded overhang
producible by the type IIs restriction endonuclease recognising the
recognition site (i) of an entry DNA other than 1, such as 2 or 3.
Such destination vectors are depicted in FIG. 4.
[0188] For inserting a ligation product from multiple nucleic acid
fragment constructs into the destination vector, the cleavage site
of item (III) of the destination vector may be made complementary
to the cleavage site of the entry DNA that will be linked to the
cleavage site of item (III). However, in the present invention
nucleic acid linkers may be used for this purpose, since suitable
linkers allow to link any item (iii) cleavage site to the item
(III) cleavage site of the destination vector without the need for
producing a destination vector for each possible downstream (item
(iii)) cleavage site of the entry DNAs. Since the specific item
(iii) cleavage site of an entry DNA or nucleic acid fragment
construct depends, for a given set of entry DNAs, from the number
of fragment constructs to be combined in the level 2 reaction, the
linkers provide the system with a broad applicability to many
different real life applications. Notably, a given system can be
applied to cases with different numbers of fragment constructs to
be recombined. An advantageous linker comprise in the following
order: [0189] (a) a type IIs restriction endonuclease recognition
site; [0190] (b) a cleavage site of said recognition site of item
(a); [0191] (c) a cleavage site of a further type IIs restriction
endonuclease recognition site of the following item (d); [0192] (d)
a type IIs restriction endonuclease recognition site defining the
cleavage site of item (c) and being a recognition site of a type
IIs restriction endonuclease different from that of item (a);
[0193] (e) a type IIs restriction endonuclease recognition site,
preferably of the same endonuclease as the recognition site of item
(d); [0194] (f) a cleavage site of said recognition site of item
(e); [0195] (g) a cleavage site of a further type IIs restriction
endonuclease recognition site of the following item (h); [0196] (h)
a type IIs restriction endonuclease recognition site defining the
cleavage site of item (g), preferably of the same endonuclease as
the recognition site of item (a).
[0197] The linkers may be linear DNA molecules. Generally, however,
the linkers are circular plasmids. The linkers comprise a pair of
type IIs restriction endonuclease recognition sites (items (a) and
(h)) and associated cleavage sites (items (b) and (g) at both ends
for linking a given item (iii) site with an item (III). This pair
of restriction sites is in convergent orientation, which means that
the two cleavage sites are oriented toward the center of the
linker, while the recognition sites are oriented towards the
termini of the linker so that the recognition sites are removed
upon restriction. Examples of linkers are linkers pELB-1 to -7 and
pELR-1 to -7 shown in FIG. 4.
[0198] The linkers preferably comprise a further, different, pair
of type IIs restriction sites flanked by the pair formed by items
(a), (b), (g) and (h) of the linker. This further pair is formed by
items (c) to (f) of the linker and is in divergent orientation,
which allows to reopen a level 2 reaction product produced using
such linker for insertion of further nucleic acid fragment
constructs.
[0199] The cleavage site of item (b) is complementary to an item
(iii) cleavage site of an entry DNA for being capable of linking a
cleavage site of item (iii) of one of a entry DNAs numbered 1 to n,
preferably of number 1 to n-1, to a cleavage site of item (III) of
said destination vector. The cleavage site of item (g) of the
linker is complementary to the cleavage site of item (III) of the
linker.
[0200] The linkers may be provided as part of a plasmid containing
the linker elements (a) to (h) defined above and a plasmid backbone
linking elements (a) and (h). The backbone may contain a selectable
marker for selecting cells containing the plasmid using a selective
agent. This allows storage and amplification of linkers in cells,
notably bacterial cells.
[0201] In a preferred embodiment, the system comprises a set of n
nucleic acid linkers numbered 1 to n, each n-th linker comprising
items (a) to (h), the cleavage site of item (iii) of each n-th
entry DNA is complementary to the cleavage site of item (b) of the
n-th linker; the cleavage site of item (g) of each n-th linker
being complementary to the cleavage site of item (III) of the n-th
destination vector. Thus, each n-th linker is capable of linking a
cleavage site of item (iii) of the n-th entry DNA to a cleavage
site of item (III) of each n-th destination vector. In this
embodiment, the system contains the same number of n entry DNAs and
linkers. For each entry DNA of the set of n entry DNA, a linker is
provided allowing linking the item (iii) cleavage site to the item
(III) cleavage site of the destination vector. Thus for a given
destination vector, all item (g) cleavage sites of the set of n
linkers can be identical. As an example, FIG. 4 shows a set of 7
entry vectors (level 1 destination vectors) PL1F-1 to -7 and a set
of linkers pELB-1 to -7. Each of the n linkers of the set of n
linkers may be part of a plasmid.
[0202] The cleavage sites of items (b) and (c) within each linker
may have the same sequence of nucleotides and may overlap such that
one and the same sequence of nucleotides provides the cleavage site
of items (b) and that of item (c). Similarly, the cleavage sites of
items (f) and (g) within each linker may have the same sequence of
nucleotides and may overlap such that one and the same sequence of
nucleotides provides the cleavage site of items (f) and that of
item (g).
[0203] In some embodiments, it may be desired to use a given entry
DNA of number >1 at a position 1 in the reaction product of the
level 2 reaction. For this purpose, the system of the invention may
comprise from 1 to n multiple destination vectors numbered 1 to n,
each of said 1 to n destination vectors having segments (I) to
(III) as defined above and optionally a segment (IV) as defined
above. The cleavage sites of item (III) of all destination vectors
may be identical and all cleavage sites of item (I) of all n
destination vectors may be unique among the cleavage sites of item
(III). Preferably, the n-th item (I) cleavage site of all n
destination vectors is complementary to the n-th item (i) cleavage
site of the entry DNA. An example of such embodiment is given in
FIG. 4 that shows a set of 7 level 2 destination vectors having
identical item (III) cleavage sites (GGGA) that are complementary
to the GGGA cleavage sites of the linkers. The item (I) cleavage
sites of each level 2 destination vector is complementary to the
level 1 destination vector shown in the same line in the left-most
column.
[0204] The optional insert of item (IV) of the destination
vector(s) may be any sequence linking items (III) and (I), whereby
the destination vector will be a circular molecule of vector.
Absence of the insert of item (IV) may mean that the destination
vector is linear DNA molecule. Preferably, however, an insert is
used that is or comprises a marker gene that allows to distinguish
cell clones containing the destination vector from those containing
the product of the level 2 reaction. Since the restriction sites of
the destination vector are in divergent orientation with respect to
the insert (see FIG. 4), the insert is lost in the level 2
reaction. Thus, destination vectors and level 2 reactions products
can be distinguished by the absence of red color in cell clones
containing the latter.
[0205] In the invention, entry DNAs and nucleic acid fragment
constructs differ in that the latter contain a sequence segment
(item (ii')) of the nucleic acid construct of interest to be
produced. For allowing introduction of such sequence segment with
its core portion into the entry DNA in a level 1 reaction, each
sequence portion of item (ii) of each entry DNA 1 to n generally
comprises a further pair of two type IIs restriction endonuclease
recognition sites oriented such that said further pair of
recognition sites can be removed from said entry DNAs by treatment
with type IIs restriction endonuclease(s) recognising said further
pair of recognition sites. In FIG. 4, the further pair of two
recognition sites are the BsaI sites in the level 1 destination
vectors. The sequence region of item (ii) is not particularly
restricted and may be as short as a few nucleotides. In one
embodiment, however, the sequence region of item (ii) contains a
reporter gene or reporter genes allowing color selection of cell
clones containing the reporter gene(s). Said further pair of
recognition sites may flank the reporter gene for enabling
selection of cell clones for the presence or absence of said
reporter gene e.g. by color. In FIG. 4, the reporter gene is lacZ
flanked by a pair of BsaI sites. The further pair of two type IIs
restriction endonuclease recognition sites are recognition sites of
endonucleases different from those of recognition sites of item (i)
and item (iii) so that a given endonuclease does not cleave a
cleavage site of the further pair and, at the same time, a
recognition site of item (i) or (iii). The orientation of the
further pair of recognition sites is divergent with respect to the
marker gene, so that the reporter gene and the recognition sites is
removed upon treatment with the type IIs endonuclease recognising
these site, so that these recognition sites are not present in the
nucleic acid fragment construct obtained in the level 1
reaction.
[0206] The second type of reactants of the level 1 reaction is one
or more modules that can be incorporated into the entry DNAs in the
level 1 reaction using the known methodology described in Engler et
al. PLoS ONE 4 (2009) e5553. These modules are also referred to
herein as "level 0 modules", since they can be produced in a level
0 reaction. An example of a level 1 reaction is schematically shown
in FIG. 5A. One or more level 0 modules are ligated together in a
desired order with and into the entry DNA to produce the level 1
fragment constructs (level 1 construct), using the inner pair of
type IIs restriction sites present in sequence portion of item (ii)
of the entry DNA. This leaves the recognition and cleavage sites of
items (i) and (iii) unchanged, whereby these are also present in
the reaction product for use in the subsequent level 2 reaction.
Since at least 3 nucleic acid fragment constructs are employed in
method of the invention, at least 3 level 1 reactions are typically
performed separately. Multiple level 1 constructs and a level 2
destination vector are then combined in a one pot reaction on level
2.
[0207] In the method of the invention, a nucleic acid construct of
interest is produced from at least m nucleic acid fragment
constructs numbered 1 to m. Each nucleic acid construct of interest
typically comprises a sequence segment to be incorporated into the
nucleic acid construct of interest. These sequence segments may be
numbered 1 to m as the nucleic acid fragment construct containing
them in the order of occurrence in the nucleic acid construct of
interest. Numeral m is an integer of at least 3, preferably at
least 6, more preferably at least 10. Said method comprises the
steps (A) to (C) as described in the following.
[0208] In step (A), the m nucleic acid fragment constructs are
provided. Each of said m nucleic acid fragment constructs
comprising in this order: [0209] (i') a type IIs restriction
endonuclease recognition site of the upstream cleavage site of item
(ii'); [0210] (ii') a sequence segment of said nucleic acid
construct of interest, said sequence segment comprising an upstream
cleavage site of the recognition site of item (i'), a core portion
of the sequence segment, and a downstream cleavage site of the
recognition site of the following item (iii'), and [0211] (iii')
the type IIs restriction endonuclease recognition site of said
downstream cleavage site of item (ii').
[0212] The m nucleic acid fragment constructs can be provided in
(separate) level 1 reactions using the entry DNAs of the system of
the invention and at least one module per type of fragment
construct that provides the core portion to the sequence segment of
item (ii'). In the level 1 reaction one or several such modules may
be combined to generate the fragment constructs with the desired
core portion comprising portions derived from multiple modules. The
modules used in the level 1 reaction are also referred to herein as
"level 0 modules", as they can be prepared in a restriction and
ligation step before the level 1 reaction. The level 1 reaction may
be performed as explained with reference to FIGS. 7 and 8 using
known methods and as described herein. It is also possible to use
more than module having identical type IIs restriction endonuclease
cleavage sites in one restriction and ligation reaction, allowing
the generation of libraries of nucleic acid fragment constructs.
For example, five promoter modules P1 to P5 as depicted in FIG. 7
may be combined in one reaction, whereby a mixture of level 1
fragment constructs is obtained that differ by having different
promoters. Thus, the mixture of fragment constructs may be screened
for the most suitable promoter function in the context of the
remaining modules introduced into the fragment constructs.
Similarly, libraries of fragment constructs containing different
5'UTRs, signal peptides, ORFs, terminators, combinations thereof or
other elements may be produced and screened for a suitable property
using the invention.
[0213] The downstream cleavage sites of nucleic acid fragment
constructs 1 to m-1 are complementary to the upstream cleavage
sites of nucleic acid fragment constructs 2 to m, respectively, for
allowing assembly of the nucleic acid fragment constructs in the
order corresponding to the numbering of the constructs in the
subsequent step (B). In the nucleic acid fragment constructs, the
recognition sites of items (i') and (iii') as well as the upstream
and downstream cleavage sites of item (ii') are derived from the
entry DNAs used, whereas the core portion is essentially derived
from the level 0 modules.
[0214] If the nucleic acid fragment constructs are provided in a
level 1 reaction, the products of the level 1 reaction are
generally transformed into cells for amplification and
purification. Typically, they are transformed into competent
bacterial cells such as E. coli cells. After cell growth, the
fragment constructs are isolated from the cells, e.g. using
standard plasmid preparation protocols, for use in the following
step (B).
[0215] The method of the invention comprises two steps wherein
fragment constructs are combined, namely the following steps (B)
and (C). In these steps, at least one fragment construct is used in
step (C) that is derived from the same entry DNA as a fragment
construct used in step (B). Thus, the method of the invention
allows reuse of entry DNAs for more than one nucleic acid fragment.
It is an important aspect of the invention that many different
fragment constructs can be combined with a relatively small number
entry DNAs. However, in this embodiment, fragment constructs
derived from the same entry DNA have the same upstream and
downstream cleavage sites (ii') and are therefore used in separate
reactions to avoid statistical inclusion of either fragment
construct at a selected position into the final nucleic acid
construct of interest.
[0216] For this purpose, the downstream cleavage site of a nucleic
acid fragment construct u, wherein u is an integer that is <m
and at least 2, is complementary to the upstream cleavage site of
the type IIs restriction endonuclease recognition site of item
(ii') of nucleic acid fragment 1 (illustrated in FIG. 4 by the long
dashed arrow linking cleavage sites TGCC of pL1F-1 and pL1F-7).
[0217] Step (B) is a level 2 reaction. In the terminology used with
reference to the figures, step (B) is a level 2i-1 reaction. In
step (B), the sequence segment(s) of item (ii') of nucleic acid
fragment constructs 1 to s, wherein s is an integer <u, and said
linker are ligated, in this order, and inserted into said
destination vector. This may be done by reacting, in the presence
of a type IIs restriction endonuclease recognising said type IIs
restriction endonuclease recognition sites of items (i') and (iii')
and items (I) and (III) of the destination vector defined below and
in the presence a DNA ligase, in reaction medium compatible with
activity of said type IIs restriction endonuclease and said ligase.
For example, a mixture comprising nucleic acid fragment constructs
1 to s, the destination vector and a linker may be treated with the
type IIs restriction endonuclease and the DNA ligase in a reaction
medium compatible with activity of the type IIs restriction
endonuclease and the ligase. Thus, s defines the number of nucleic
acid fragment constructs combined in step (B) with the (level 2)
destination vector. Since s is smaller than u, nucleic acid
fragment construct u+1 and higher will not be used in step (B), but
in a subsequent step such as step (C).
[0218] The linker that may be used in step (B) is as defined above.
Cleavage site (b) of the linker may be complementary to the
downstream cleavage site of item (ii') of nucleic acid fragment
construct s, and cleavage site (g) of said linker and the cleavage
site of item (III) of the destination vector may complementary for
connecting the downstream cleavage site of fragment construct s to
site (III) of the destination vector.
[0219] Step (B) may comprise transformation of the restriction and
ligation product into cells for amplification and purification.
Typically, it is transformed into competent bacterial cells such as
E. coli cells. After cell growth, the level 2 construct is
generally isolated from the cells, e.g. using standard plasmid
preparation protocols, for use in the following step (C).
[0220] Step (C) is a subsequent level 2 reaction. In the
terminology used with reference to the figures, step (C) is a level
2-2 or level 2i-2 reaction. In step (C), a mixture comprising the
recombination product of step (B) (a "level 2i-1 construct") and
nucleic acid fragment construct(s) s+1 to m is treated with a type
IIs restriction endonuclease recognising said type IIs restriction
endonuclease recognition sites of items (i') and (iii'), a type IIs
restriction endonuclease recognising said type IIs restriction
endonuclease recognition sites of items (d) and (e) of the linker
and a DNA ligase in a reaction medium compatible with activity of
said type IIs restriction endonucleases and said ligase. Thereby,
the sequence segments of item (ii') of nucleic acid fragment
constructs s+1 to m and optionally a further linker as defined in
item (3) are inserted into the cleavage sites provided by items (c)
and (f) of the linker used in step (B). The recognition sites of
items (i') and (iii') may be the same as the recognition sites of
items (d) and (e) of the linker, whereby a type IIs restriction
endonuclease recognising all these recognition sites can be used.
The linker may be of the type pELE shown in FIG. 4, whereby no
further level 2 reaction can be performed with the reaction product
of step (C). Alternatively, a linker as defined in item (3) may be
used, whereby a further level 2 reaction can be conducted. Thus,
nucleic acid constructs of interest can be made from more than m
nucleic acid fragment constructs. The possibility to use one or
more further level 2 reactions is schematically shown in FIG.
6.
[0221] Step (C) may comprise transformation of the restriction and
ligation product into cells for amplification and purification.
Typically, it is transformed into competent bacterial cells such as
E. coli cells. After cell growth, the construct of step (C) may be
isolated from the cells, e.g. using standard plasmid preparation
protocols.
[0222] The present invention provides a further system for
producing a nucleic acid construct of interest as defined in claims
16 to 24. Similar as with the system described above, consecutive
repetitions of cloning steps and re-use of the cleavage sites from
a predefined set of vectors allows to increase the number of
fragments that make up a nucleic acid construct of interest in a
vector. In this system, a set of n destination vectors is used that
are referred to as "level M destination vectors". Level M
destination vectors differ from level 2 destination vectors in that
an additional type IIs restriction endonuclease recognition site is
present (compare the level 2 destination vectors of FIG. 4 with the
"level M destination vectors" in FIG. 31). The additional
recognition site has a cleavage site of the same nucleotide
sequence as the cleavage site of item (I) of the level 2
destination vectors. Fragment constructs or entry DNAs are inserted
into the level M destination vectors together with linkers referred
to as "linkers M". Linkers M differ from linkers such as linkers
pELE shown in FIG. 4 in that they have an additional type IIs
restriction endonuclease recognition site (compare linkers pELE of
FIG. 4 with the "end liners M" in FIG. 31). The additional
recognition site of linkers M has a cleavage site of the same
nucleotide sequence as the cleavage site on the left hand side of
linkers pELE of FIG. 4. The additional recognition sites of
destination vectors M and linkers M allow excision of constructs
cloned into the designation vectors M and introduction, preferably
with other constructs produced in parallel level M reactions, into
a further destination vector referred to as "level P destination
vector" together with a suitable linker referred to as linker P.
Similarly as destination vectors M and linkers M, destination
vectors P and linkers P are designed such that excision of
constructs cloned into the designation vectors P is possible as
well as reintroduction into a further level M destination vector.
Since in each cloning step, multiple fragment constructs prepared
in parallel preceding steps can be combined, the number of fragment
constructs combined into a construct of interest can be increased
multiplicatively, which is indicated by letter M in "destination
vector M". In any event, a set of n destination vectors M and a set
of n linkers M, preferably in combination with a set of n
destination vectors P and set of n linkers P, allows reuse of a
limited number of n cleavage sites such that a large number of
fragment constructs (90 in FIG. 36) can be assembled with a small
number of elements n in said sets.
[0223] n is at least 2, preferably at least 3, more preferably at
least 4. The versatility of the system increases with increasing n.
However, it is not necessary to have n>10. Thus, n may be a
number of from 3 to 20, preferably of from 4 to 10, more preferably
of from 5 to 9 or from 6 to 8. In the figures, embodiments with n=7
are exemplified, which is the most preferred embodiment.
[0224] The cleavage sites of items (II') and (III') of destination
vectors M may overlap completely. In this case, one physical
sequence of nucleotides provides the cleavage sites of two
different type IIs restriction endonuclease recognition sites.
Analogously, one physical sequence of nucleotides may provide the
cleavage sites of two different type IIs restriction endonuclease
recognition sites, namely the cleavage sites of items (b') and
(c'), of items (II') and (III') and of items (b'') and (c''). This
embodiment is used in the examples shown in the figures. However,
it is also possible that the cleavage sites of the pairs mentioned
before are adjacent separated cleavage sites.
[0225] The number of entry DNAs to be used in not decisive in the
system of this embodiment. It is possible that one entry DNA is
incorporated into a level M destination vector, optionally followed
by incorporation of one level 1 construct into a level P
destination vector. However, the main advantages of the system can
be made use of if at least 2, at least 3, at least 4, or at least 5
entry DNAs are combined by introduction into a destination vector
M. The recognition sites of items (i), (iii), (I') and (VII'),
(a'), and (f') may be recognition sites of the same type IIs
restriction endonuclease.
[0226] Multiple level M reactions can be conducted in parallel in
separate reaction vessels as indicated in FIG. 32a and b. The
separate level M constructs may be combined in a subsequent level 2
or level P reaction. The destination vector M used in the second
level M reaction is selected such that it has a complementary
cleavage site to the cleavage site provided by the linker M used in
the first level M reaction. In this way, two or more level M
constructs can be combined in a level 2 reaction (FIG. 32c) or a
level P reaction (FIG. 34a). In FIG. 32c, it is the TTAC cleavage
site in the level M construct 1 that is derived from a linker M
used in a first level M reaction. The TTAC cleavage site in the
level M construct 2 is derived from a destination vector M used in
the second level M reaction. Cleavage by BsaI allows ligation of
the construct comprising TU1 to TU3 with the construct comprising
TU4 and TU5 into the destination vector level 2. Alternatively, as
shown in FIG. 34a, a destination vector P and linker P can be
treated with BsaI and ligase together with level M constructs 1 and
2 to give the level P construct shown at the bottom of FIG.
34a.
[0227] In the set of n destination vectors P, the same set of
cleavage sites is used as in the destination vectors M. The
cleavage sites (VI'') of all n destination vectors P are identical
and are at the same time identical to the cleavage sites of items
(VI') of destination vectors M. In the set of n linkers P, the same
set of cleavage sites is used as in the linkers M and the
destination vectors M. Thus, a limited set of n cleavage sites
allows to combine a number of fragments constructs that can far
exceed the number of n. As shown in FIG. 34b, multiple level P
constructs can be combined into another destination vector M using
a linker M.
[0228] The recognition sites of items (I''), (IV'), (d'), (a'') and
(f'') may be recognition sites of the same type IIs restriction
endonuclease, and the recognition sites of items (IV''), (I'),
(VII'), (a') and (f') may be recognition sites of the same type IIs
restriction endonuclease.
[0229] Item (VIII') of destination vector M may have a marker
allowing selection of ligation product of a level M reaction for
absence of item (VIII'). The marker may be lacZ for blue/white
selection. Item (VIII'') of destination vector P may have a marker
allowing selection of ligation product of a level P reaction for
absence of item (VIII''). Generally, the designation vectors and
the linkers are circular molecules or plasmids containing a
selectable marker in their backbone.
[0230] An advantage of systems and methods of the invention is that
cloning steps can be done as one-pot reactions, requiring only a
simple incubation such as in a thermocycler. In particular, this
avoids the need for labor-intensive and operations that are
difficult to automate such as purification of DNA fragments from
agarose gels. This mean that all the elements required for the
design of a completely automatized cloning system are now in place.
Operations that are employed are preparation of miniprep DNA,
liquid handling and incubation to perform restriction-ligation,
plating of transformations on plates, picking of colonies, and
digestion and analysis of miniprep DNA. This last step may be
replaced by DNA sequencing, as very few colonies need to be
screened to obtain the desired construct (in the majority of cases,
one colony is sufficient). All these operations can easily be
handled by standard automation robots. This aspect promises to
revolutionize the number of constructs that can be made within a
given time as well as the production costs for these constructs,
and therefore opens the door for new applications that will be
required in the field of synthetic biology.
[0231] Another advantage of the invention compared to more
traditional cloning strategies is that complex design of specific
construction strategies is not needed anymore, since the design is
automatically defined by the number and the order of modules (or
genes) that a user wants to assemble. The cloning strategy in fact
can be easily and unambiguously determined by a simple computer
program. This program can be directly linked to the automation
robots that would physically make the construct. An advantage of
such system is that the cloning strategy itself cannot become a
limiting factor when constructs reach a large size, since the same
principles and the same cloning vectors can be reused indefinitely.
In fact, it is conceivable that the invention can be used to clone
entire chromosomes.
DETAILED DESCRIPTION OF THE INVENTION
[0232] The system of the invention allows the production of nucleic
acid constructs of interest from multiple nucleic acid fragments
constructs using a combination of nucleic acid fragment constructs
via single-stranded overhangs formed at both ends of the fragments
using type IIs restriction endonucleases. In the invention, type
IIs restriction enzymes are used. The type IIs restriction
endonuclease recognition site is a recognition site of a
restriction endonuclease recognizing a double-stranded DNA and
cleaving the double-stranded DNA at a cleavage site that is outside
the recognition site on the double stranded DNA. The type IIs
restriction endonuclease cleaves such that, depending on the
specific type IIs restriction endonuclease, overhangs of from 3 to
6 nucleotides are produced. However, it is also possible to use
type IIs endonucleases producing longer single-stranded overhangs.
The nucleotide range that forms the overhangs upon cleavage is
referred to herein as cleavage site. Since the nucleotides of the
cleavage site are not part of the recognition site, they can be
chosen as desired without destroying cleavage activity of the type
IIs restriction endonuclease. Examples of type IIs restriction
endonucleases suitable for the methods of the invention are given
below.
[0233] For practicing the invention, not only BsaI and BpiI, but
any type IIs restriction enzyme that provides "sticky" ends
sufficient for efficient ligation at its cleavage sites can be
used. A selection of such enzymes is provided on the REBASE webpage
(rebase.neb.com/cqi-bin/asvmmlist) and in the review of Szybalsky
et al. (1991, Gene, 100:13-26). Type II restriction enzymes with
asymmetric recognition sites (e.g. those shown in this webpage)
that have cleavage site outside of recognition site and provide
upon cleavage of at least three, preferably 4 or more nucleotide
residues overhangs (e.g. Bli7361; BpuAl, VpaK321, SfaNI, etc.) can
be used in the invention. It is recommended that the recognition
site contains at least 4, more preferably at least 6 or more base
pairs in order to minimize the chance for such site to be found in
a sequence portion of interest. Type IIs restriction nucleases with
5 bp recognition sites (e.g. SfaNI) also can be used. Type IIs
restriction endonucleases that produce 4 nt single-stranded
overhangs at the extremities of digested fragments can
theoretically generate ends with 256 possible sequences. Type IIs
restriction enzymes having even longer recognition sites, e.g.
comprising ten or more base pairs have been engineered. The largest
recognition site among natural type IIs enzymes is for the enzyme
SapI which has a 7 bp recognition site. A preferred solution is the
use of artificial type IIs enzymes engineered to have a long
recognition site (Lippow et al, 2009, Nucleic acides Res.,
37:3061-3073). For example, a type IIs enzyme with a 18 bp
recognition sites would be expected to cut only a few times per
eukaryotic genome at most, and would allow to make most entry
modules without having to change any nucleotide of the native
sequence.
TABLE-US-00001 TABLE 1 List of usable type IIs restriction enzymes
commercially available reach reach Recognition top bottom exten-
Name sequence strand strand sion BsaXI (9/12) 10 7 3 nt 3'
ACNNNNNCTCC (10/7) Bst6I CTCTTC (1/4) 1 4 3 nt 5' Eam1104I CTCTTC
(1/4) 1 4 3 nt 5' EarI CTCTTC (1/4) 1 4 3 nt 5' LguI GCTCTTC (1/4)
1 4 3 nt 5' PciSI GCTCTTC (1/4) 1 4 3 nt 5' BspQI GCTCTTC (1/4) 1 4
3 nt 5' SapI GCTCTTC (1/4) 1 4 3 nt 5' BveI ACCTGC (4/8) 4 8 4 nt
5' Acc36I ACCTGC (4/8) 4 8 4 nt 5' BfuAI ACCTGC (4/8) 4 8 4 nt 5'
BspMI ACCTGC (4/8) 4 8 4 nt 5' AarI CACCTGC (4/8) 4 8 4 nt 5' Esp3I
CGTCTC (1/5) 1 5 4 nt 5' BsmBI CGTCTC (1/5) 1 5 4 nt 5' BstV2I
GAAGAC (2/6) 2 6 4 nt 5' BpiI GAAGAC (2/6) 2 6 4 nt 5' BpuAI GAAGAC
(2/6) 2 6 4 nt 5' BbsI GAAGAC (2/6) 2 6 4 nt 5' BseXI GCAGC (8/12)
8 12 4 nt 5' Lsp1109I GCAGC (8/12) 8 12 4 nt 5' BstV1I GCAGC (8/12)
8 12 4 nt 5' BbvI GCAGC (8/12) 8 12 4 nt 5' SfaNI GCATC (5/9) 5 9 4
nt 5' LweI GCATC (5/9) 5 9 4 nt 5' BtgZI GCGATG (10/14) 10 14 4 nt
5' FokI GGATG (9/13) 9 13 4 nt 5' FaqI GGGAC (10/14) 10 14 4 nt 5'
BslFI GGGAC (10/14) 10 14 4 nt 5' BsmFI GGGAC (10/14) 10 14 4 nt 5'
Bso31I GGTCTC (1/5) 1 5 4 nt 5' BspTNI GGTCTC (1/5) 1 5 4 nt 5'
Eco31I GGTCTC (1/5) 1 5 4 nt 5' BsaI GGTCTC (1/5) 1 5 4 nt 5'
Alw26I GTCTC (1/5) 1 5 4 nt 5' BstMAI GTCTC (1/5) 1 5 4 nt 5' BsmAI
GTCTC (1/5) 1 5 4 nt 5' BaeI (10/15) 12 7 5 nt 3' ACNNNNGTAYC
(12/7) PpiI (7/12) 13 8 5 nt 3' GAACNNNNNCTC (13/8) PsrI (7/12) 12
7 5 nt 3' GAACNNNNNNTAC (12/7) AloI (7/12) 12 7 5 nt 3'
GAACNNNNNNTCC (12/7) BarI (7/12) 12 7 5 nt 3' GAAGNNNNNNTAC (12/7)
AjuI (7/12) 11 6 5 nt 3' GAANNNNNNNTTGG (11/6) TstI (8/13) 12 7 5
nt 3' CACNNNNNNTCC (12/7) Hin4I (8/13) 13 8 5 nt 3' GAYNNNNNVTC
(13/8) HgaI GACGC (5/10) 5 10 5 nt 5' CseI GACGC (5/10) 5 10 5 nt
5'
TABLE-US-00002 TABLE 2 Preferred type IIs restriction enzymes reach
reach Recognition top bottom exten- Name sequence strand strand
sion LguI GCTCTTC (1/4) 1 4 3 nt 5' PciSI GCTCTTC (1/4) 1 4 3 nt 5'
BspQI GCTCTTC (1/4) 1 4 3 nt 5' SapI GCTCTTC (1/4) 1 4 3 nt 5' BveI
ACCTGC (4/8) 4 8 4 nt 5' Acc36I ACCTGC (4/8) 4 8 4 nt 5' BfuAI
ACCTGC (4/8) 4 8 4 nt 5' BspMI ACCTGC (4/8) 4 8 4 nt 5' AarI
CACCTGC (4/8) 4 8 4 nt 5' Esp3I CGTCTC (1/5) 1 5 4 nt 5' BsmBI
CGTCTC (1/5) 1 5 4 nt 5' BstV2I GAAGAC (2/6) 2 6 4 nt 5' BpiI
GAAGAC (2/6) 2 6 4 nt 5' BpuAI GAAGAC (2/6) 2 6 4 nt 5' BbsI GAAGAC
(2/6) 2 6 4 nt 5' BtgZI GCGATG (10/14) 10 14 4 nt 5' Bso31I GGTCTC
(1/5) 1 5 4 nt 5' BspTNI GGTCTC (1/5) 1 5 4 nt 5' Eco31I GGTCTC
(1/5) 1 5 4 nt 5' BsaI GGTCTC (1/5) 1 5 4 nt 5' HgaI GACGC (5/10) 5
10 5 nt 5' CseI GACGC (5/10) 5 10 5 nt 5'
[0234] Most preferred are the following type IIs restriction
endonucleases: SapI, BspMI, AarI, Esp3I, BpiI, BsaI and HgaI. Many
of the cited restriction endonucleases are available from New
England Biolabs. Sources of these enzymes can also be found on the
REBASE webpage mentioned above.
[0235] Examples of ligases to be used in the invention include T4
DNA ligase, E. coli DNA ligase, Taq DNA ligase, all of which are
commercially available from New England Biolabs.
[0236] In the following, the invention will be further described
with reference to specific embodiments, examples and the
figures.
[0237] FIG. 1a shows elements of a system that allows re-use of the
entry DNAs of the invention (level 1 destination vectors) for
different inserted sequence segments. This system comprises:
(1) n nucleic acid fragment constructs ("na", shown for n=1 to 7),
each flanked by two sequences Sx and Sy representing cleavage sites
of a type IIs restriction endonuclease. After restriction
endonuclease digestion, the cleavage sites form single-stranded
overhangs that are complementary from one nucleic acid fragment
constructs (as well as the underlying entry vector) to the next,
which is indicated by the same index of "S". The cleavage site at
the 3' end (right hand side in the figures) of the last construct
(na7) forms a single-stranded overhang compatible with the overhang
created by cleavage of the cleavage site at the 5' end (left hand
side in the figures) of the first fragment construct na1 by
restriction endonuclease digestion, as indicated by the same
numbering "S1" at these sites; (2) a set of n `end-linkers` (ELx, x
indicating the numbering from 1 to 7) flanked on one side (5' end)
with a cleavage site compatible with the 3' cleavage sites (S1 to
S7) of the nucleic acid fragment constructs (as well as the
underlying entry DNA) and on the other side (3' end) with a unique
site not compatible with any of the n entry DNAs (S8); (3) a
destination vector with two cleavage sites, one site compatible
with sites S1 (or S2, S3, S4, S5, S6, S7), and the other site
compatible with cleavage site S8 of the end-linkers.
[0238] FIG. 1b provides an example for cloning of three nucleic
acid fragment constructs into a destination vector. Cloning of the
three nucleic acids fragment constructs employs ligation of the
appropriate end-linker (end-linker 3). The resulting construct can
be later re-opened at cleavage sites S4 and S8 by digestion with
the appropriate type IIs endonuclease. All other sites lack a
flanking type IIs endonuclease recognition site in the reaction
product and are thus protected from digestion with the endonuclease
used for the production of this reaction product.
[0239] FIG. 1C shows that further nucleic acid fragment constructs
na4 to na7 and a further construct na1 that is based on the same
entry vector number 1 as na1 ligated in FIG. 1b can be cloned into
the product vector obtained in FIG. 1 b. At each successive cloning
step, a different end-linker is used (ELx or ELx-b that differ by
containing different internal type IIs restriction site for
reopening the construct at the next stage). The structure of the
end-linkerse will be more specifically explained in the following
figures).
[0240] FIG. 2 explains how type IIs restriction sites are depicted
in the following figures. A type IIs restriction endonulease site
contains a recognition sequence (also referred to herein as
"recognition site") and a cleavage site located outside of the
recognition sequence. The nucleotide sequence of the cleavage site
is shown in a horizontally elongated box. The recognition site can
be found on either side of the cleavage site, depending on the
orientation of the asymmetrical recognition site in the DNA. When
the recognition sequence is located on the left of the cleavage
site, the recognition site is illustrated as a vertically elongated
box on the left half under the box representing the cleavage site.
When the recognition sequence is located on the right of the
cleavage site, the recognition site is illustrated as a vertically
extended box on the right half under the box representing the
cleavage site. The nucleotide sequences of the top DNA strand are
shown next to the represented type IIs restriction sites. In the
third and fourth row, two restriction sites are shown the cleavage
sites of which are oriented towards each other, but are non
overlapping and separated by an "optional sequence". In the fifth
and sixth row, two restriction sites are shown the cleavage site of
which overlap over the entire range of 4 base pairs.
After cloning using a type IIs enzyme, the corresponding
recognition site is usually eliminated during cloning. If
recognition sites on both sides of the cleavage site are
eliminated, only the sequence of the cleavage site (4 bases in the
examples depicted) is left in the DNA as shown schematically at the
bottom. FIG. 3 shows a general embodiment wherein a eukaryotic
multi-gene construct is produced as the nucleic acid fragment of
interest. FIG. 3 illustrates a general strategy that can be used.
Basic genetic elements such as promoters, 5' untranslated regions
(5' UTRs), signal peptides (SP), open reading frames (ORFs), and
terminators (T) are cloned as basic modules ("level 0 modules").
Libraries of each of these types of genetic elements may be
provided as shown at the top. The libraries of `level 0` entry
modules may be stored until needed for cloning. When a construct
needs to be made, genetic elements cloned as level 0 modules are
chosen (second row in the FIG. 3), and are assembled, in a level 1
reaction, using a one-pot Golden Gate cloning reaction into an
entry DNA. The level 1 reaction product (schematically shown in the
third row), referred herein as nucleic acid fragment construct,
contains an assembled transcription unit comprising the chosen
elements in the desired order. In a second step of cloning, two or
more (11 in the depicted example) different transcription units
(that can be made in separate level 1 reactions) are assembled in a
desired order into a destination vector (level 2) to obtain a level
2 multigene construct of interest.
[0241] At the bottom, the basic gene structures for secreted and
cytosolic proteins are shown. Since the latter have no signal
peptide (SP), the ORF level 0 modules for cytosolic proteins may
have the cleavage site sequences of the signal peptides used for
secreted proteins for allowing linking of the ORF module with the
3' end of the module for the 5' UTR in the level 1 reaction.
[0242] FIG. 4 depicts a set of level 1 destination vectors, level 2
destination vectors, and end-linkers. The level 1 destination
vectors and end-linkers are cloned in plasmids with a carbenicillin
resistance selectable marker, while level 2 destination vectors
carry a kanamycin resistance gene. Other antibiotic resistance
genes could also be used instead of the ones used in this example.
Level 1 destination vectors of the series pL1F-1 to -7 and of the
series pL1R-1 to -7 differ by the cleavage sites of the internal
pair type IIs restriction sites (the BsaI sites). The sites of both
series are designed for cloning of the same assembled transcription
units, but in opposite orientation. The level 1 destination vectors
have an internal lacZ reporter gene that is removed together with
the recognition sites of the internal pair of type IIs restriction
sites (the BsaI sites) in the level 1 reaction due to the divergent
orientation of these sites with respect to LacZ. Thus, lacZ is not
present in the level 1 reaction product, allowing blue/while
selection of cell clones containing the reaction product. The outer
restriction sites in the level 1 destination vectors (the BpiI
sites) are in convergent orientation with respect to the BsaI sites
and lacZ and are unchanged in the level 1 reaction, since the
restriction endonuclease of the inner pair of restriction sites
(BsaI), but not the restriction endonuclease of the outer pair of
restriction sites (BpiI), is used in the level 1 reaction. Straight
dashed arrows indicate the complementarity of the cleavages sites
of the right-hand BpiI sites of each of entry DNAs 1 to 6 with the
left-hand entry DNA directly underneath which allows the ligation
of entry DNAs 1 to 7, as well as the level 1 constructs derived
therefrom, in the level 2 reaction via complementary
single-stranded overhangs produced by BpiI digestion.
Complementarity of the right-hand cleavage site of the bottom entry
DNA (of sequence TGCC) with the left-hand cleavage site of the
first entry DNA allows to reuse the entry DNA 1 and the following
entry DNAs in a second level 2 reaction. In a second level 2
reaction, the reused entry DNAs may be provided in a level 1
reaction with a different insert compared to the insert (referred
to as "core portion of the sequence segment" in item (ii') of the
method of the invention). Thus, more nucleic acid fragment
constructs can be combined into a nucleic acid construct of
interest than the number of elements of the set of entry DNAs.
"Divergent" herein means that the two cleavage sites of a pair of
restriction sites are more remote than the recognition sites from a
position between the two restriction sites. "Convergent" herein
means that the two recognition sites of a pair of restriction sites
are more remote than the cleavages sites from a position between
the two restriction sites.
[0243] The set of level 2 destination vectors shown has the same
number of elements as the number of level 1 destination vectors.
The level 2 destination vectors have a pair of divergent (with
respect to the central portion in which nucleic acid fragment
constructs are inserted in the level 2 reaction) type IIs
restriction sites flanking genes ("CRed") providing a red
phenotype. For each upstream BpiI cleavage site of the entry DNAs
there is a level 2 destination vector having a complementary
upstream BpiI site. Thus, each entry DNA can be used to produce a
nucleic acid fragment construct that will take position 1 in the
level 2 reaction product.
[0244] Three sets of end-linkers are depicted, each set generally
having the same number of elements as the number of level 1 and
level 2 destination vectors. Sets pELB and pELR are similar in that
they have the same cleavage sites and outer recognition sites. Sets
pELB and pELR both have a further inner recognition site that will
be unchanged in the level 2 reaction, whereby they are present in
the level 2 reaction product. Thus, they can be used for inserting,
in a second or further level 2 reaction, further nucleic acid
fragment constructs into the reaction product of the first level 2
reaction. This is not possible if an end-linker from the pELE set
is used, since these lack the inner divergent pair of restriction
sites. Sets pELB and pELR differ in that different inner
recognition sites (BsaI versus Esp3I) are used and in that
different central reporter genes for color selection of cell clones
are used. All end-linkers can be used for joining the nucleic acid
fragment constructs derived from the level 1 destination vectors to
the downstream cleavage site of the level 2 destination vectors
using cleavage site GGGA. Thus all destination vectors and all
end-linkers have the same downstream cleavage site (GGGA). For each
downstream BpiI cleavage site of the entry DNAs there is a linker
having a complementary upstream BpiI cleavage site.
[0245] FIG. 5 illustrates the structure of the modules depicted in
FIG. 3 in greater detail and shows how these modules can be
assembled. At each cloning step, constructs can be assembled by
mixing in one tube all constructs or DNA fragments required,
together with the appropriate type IIS enzyme (indicated above the
horizontal reaction arrows) and ligase, and incubating the mix
under conditions allowing restriction enzyme digestion and
ligation.
[0246] Level 0 modules have an insert of interest (for example a
promoter sequence, P1) located between two convergent type IIs
restriction sites (BsaI in the example shown). Level 0 modules can
be cloned by a number of different procedures, and one example is
shown here, starting from either PCR products or level-1 constructs
(top row of the figure designated "level 0"). In this example,
cloning is performed using the enzyme BpiI in a compatible level 0
destination vector. Methods for such cloning are known from the
literature, see e.g. Engler et al. PLoS ONE 4 (2009) e5553.
[0247] Compatible sets of level 0 modules are then assembled and
cloned on level 1 into a level 1 destination vector using a Golden
Gate cloning reaction with a second type IIs enzyme, here BsaI. The
resulting level 1 constructs contain, for example, assembled
transcriptional units (TUs).
[0248] Several level 1 constructs (in the present example, 2 such
constructs indicated by "TU1" and "TU2") are then assembled
together with a selected end-linker (pELE-2, see FIG. 4) in a
compatible level 2 destination vector (pL2-1 for example). As
discussed for FIG. 1a, both level 1 constructs have to be
compatible for level 2 assembly, i.e. having convergent terminal
cleavage sites. The first level 1 construct corresponds to position
1 (the level 0 modules were cloned in the level 1 destination
vector pL1F-1 depicted in FIG. 4) and the second one corresponds to
the next position (position 2), the level 0 modules of which were
cloned in the level 1 destination vector pL1F-2). Since pELE-2 does
not contain internal type IIs restriction sites, no further nucleic
acids can be cloned in the resulting level 2 construct. In such
case, the level reaction is referred to as "level 2-1" and the
reaction product is referred to as "level 2-1 construct").
[0249] A similar level 2 reaction can however be made using
end-linker pELB-2 rather than pELE-2 (see FIG. 4). Since this
end-linker contains a pair of further internal type IIs restriction
sites (BsaI), the resulting construct ("level 2i-1 construct") will
also contain such site, and therefore can be used again as level 2
destination vector for a second level 2 reaction for insertion of
additional transcriptional units or other fragment constructs.
[0250] ++indicates that only one of several entry clones was drawn
due to space limitation. Each cleavage site is shown as a box with
the 4 nucleotides of the cleavage site; the two boxes below show
which type IIS recognition sites flank the recombination sites on
the left and right sides. P1-a/b stands for promoter fragment 1 or
2; UTR1 stands for 5' untranslated sequence; T1 indicates a
terminator; CRed stands for a red color visual marker encoding
canthaxanthin biosynthetic genes.
[0251] FIG. 5B show nucleotide sequences at the junction of various
modules for each step of cloning of FIG. 5A. "Gene 1" is equivalent
to "TU 1".
[0252] FIG. 6 is an overview of alternative cloning strategies.
Every cloning step relies on three elements that are different from
one level to the next: antibiotic selectable marker, type IIS
restriction enzyme, and visual selectable marker. Cloning after
level 2i-1 requires the use of two type IIS enzymes, such as
BpiI-BsaI or BpiI-Esp3I. Using the described set of level 1
destination vectors, level 2 destination vectors and end linkers,
and the indefinitely repeatable cloning strategy provided, as many
transcription units can be added to a construct as desired by a
user, using as many cycles of cloning as required. Physical limits
will ultimately be encountered due to handling of very large
constructs, but such limits will not come from the cloning strategy
itself. The level 2 reactions are numbered by the numeral at
position x in level 2-x.
[0253] Level 2-x stands for a level 2 reaction producing a level 2
reaction product that cannot be used for a further level 2 reaction
due to the absence of a pair of type IIs restriction sites allowing
reopening of the level 2 reaction product (e.g. due to the use of
an end-linker of the pELE set, the last "E" indicating "end").
[0254] Level 2i-x stands for a level 2 reaction that produces a
reaction product that is an intermediate (e.g. due to the use of an
end-linker of the pELB set) and can thus be used for a further
level 2 reaction.
[0255] Each level 2i-x reaction product opens up two possibilities
for a further level 2 reaction (indicated by the branching arrows).
Depending on the use of the end-linker, the next level 2 reaction
will either lead to an end (boxed level 2-x) or will lead to a
further intermediate reaction product, allowing a still further
level 2 reaction.
[0256] FIG. 7 shows the structure of level 0 modules. All level 0
modules are flanked by two convergent BsaI sites (with respect to
the insert such as P1). Five module classes, namely promoter
modules, 5'UTR modules, signal peptide modules, ORF modules and
terminator modules are depicted. All modules of a same class are
flanked by the same cleavage sites (for promoter modules GGAG and
TACT). This design allows to clone any module of a given class into
a transcription unit using the same cloning strategy. It also
allows the use of multiple modules from a module class for
obtaining libraries of level 1 fragment constructs. Modules of
different classes are designed to be compatible for assembly in a
multi-fragment level 1 reaction, also referred to herein as "Golgen
Gate" cloning reaction, i.e., the sites joining two modules form
compatible (complementary) single-stranded overhangs after
digestion with a type IIs restriction enzyme, here BsaI. A "set"
designates a group consisting of 1 module from each of the five
module classes.
[0257] FIG. 8A explains the assembly of transcriptional units from
level 0 modules into level 1 destination vectors (entry DNAs).
Module sets 1 to 9 (each consisting of a set of level 0 modules as
shown in FIG. 7) are assembled in a level 1 destination vector.
Modules sets 1 to 7 are assembled in level 1 destination vectors
pL1F-1 to -7 (see FIG. 4), respectively. Module sets 8 and 9 (and
optional) further sets) are cloned in the same series of level 1
destination vectors pLF-1, pLF-2 etc, respectively. Thus, level 1
destination vectors pL1F-1 and -2 (and optionally further vectors)
are reused for a different module set than module sets 1 and 2. The
reaction products are level 1 constructs having flanking BpiI
restriction sites retained from the level 1 destination vectors.
These flanking restriction sites define with their associated
cleavage sites the order in which the level 1 constructs are
ligated in the subsequent level 2 reaction. Each reaction shown is
generally performed as a separate reaction.
[0258] FIG. 8B illustrates an example for the assembly of level 0
modules using a different set of level 1 destination vectors. In
this example, the restriction sites for the enzyme BpiI are
identical as in FIG. 1A, but the 4-nucleotide cleavage sites of the
restriction sites for the enzymes BsaI and BpiI overlap exactly in
each destination vector. As a consequence, the cleavage sites of
the restriction sites for BsaI are not compatible with the
previously described level 0 modules (in FIG. 7). In this
embodiment, a set of adaptors that allow joining of the level 0
modules and of the level 1 destination vectors is employed. The
resulting level 1 constructs have the same overall structure as the
constructs made in FIG. 8A: the different transcription units are
all flanked by the same compatible sets of BpiI restriction sites.
These sites are convergent in each construct, and are compatible
from one construct to the next.
[0259] FIG. 9A: assembly of 5 transcription units into a multigene
level 2 construct in a level 2 reaction. An appropriate end linker
is used for linking the right-hand side of the fifth level 1
construct to the GGGA cleavage site of the destination vector
referred to as "Destination 2". The type of end-linker used does
not allow the reaction product to be used as a starting material
for a further level 2 reaction. The term "Golden Gate" means that
reactants having type IIs restriction sites with compatible
cleavage sites are combined in desired order in a reaction
comprising restriction and ligation. The level 2 construct is shown
on the right hand side in two different representations with
different degrees of detail.
[0260] FIG. 9B: Assembly is performed with an end linker containing
internal divergent BsaI restriction sites for allowing a further
level 2 reaction in the resulting level 2 construct.
[0261] FIG. 9C shows examples of further rounds of cloning. At the
top, a short version of the reaction of FIG. 9B is shown, whereby a
level 2i-1 construct is obtained. In the next level 2 reaction, 6
transcription units (designated TU6 to TU11) are added to the level
2i-1 construct, leading to a level 2i-2 construct. In the next
step, four further TUs (designated TU12 to TU15) are added, leading
to the level 2i-3 construct. In a further step shown at the bottom,
one further TU (TU16) is added to obtain the level 2-4 construct.
At each successive cloning step, different end linkers have to be
used, that may contain either internal BsaI or Esp3I restriction
sites, or none if the final round of cloning is reached.
[0262] FIG. 10 shows the efficiency of cloning for different levels
of assembly. For level 1 and level 2-1, all minipreps analyzed
contained only correctly assembled constructs, even though multiple
fragments were assembled in a one-pot one-step reaction for each
construct. Moreover, the last level 2-1 construct was obtained by
assembly in one step of 6 transcription units and one end-linker in
one destination vector. Despite the large size and large number of
components, all minipreps analyzed contained only correct
constructs. The final construct made contains 11 transcription
units. All colonies analyzed contained correct constructs despite
the large size of the construct (34 kb),
[0263] FIGS. 11 to 17 show how the cloning system of this invention
can be used to create constructs containing one or multiple
operons. Applications include microbial strain construction for
metabolic engineering.
[0264] FIG. 11 illustrates the general strategy used for cloning of
prokaryotic operons. Basic genetic elements such as promoters, open
reading frames (ORFs), and terminators are cloned as level 0
modules. Libraries of level 0 modules or individual level 0 modules
can be stored until needed for cloning. To make a desired
construct, a selected number of genetic elements cloned as level 0
modules are chosen. Unlike for cloning of eukaryotic multigene
constructs, promoters, open reading frames and terminators are not
cloned together in a level 1 destination vector. The reason for
this design is that different operons may contain a different
number of open reading frames, preventing the design of a fixed set
of compatible cleavage sites for cloning. Rather, promoters, open
reading frames and terminators are cloned separately in different
level 1 destination vectors to obtain level 1 constructs. Rather
than serving the purpose to assemble several level 0 modules
together, cloning in level 1 destination vectors for prokaryotes
serves mainly the purpose of providing positional information for
the cloned level 1 construct for a subsequent level 2 assembly.
Complete or partial operons are then cloned from several level 1
constructs in a one-pot one-step cloning reaction on level 2.
[0265] FIG. 12 Comparison of level 1 destination vectors for
eukaryotes ("MoClo" and prokaryotes ("MoClo Pro"). Level 1
destination vectors (entry DNAs) for cloning promoters and
terminators for prokaryotic operons may be the same as level 1
destination vectors made for eukaryotes (FIG. 4). In contrast,
level 1 destination vectors made for prokaryotic ORFs (open reading
frames) have different internal BsaI sites (AATG and GCTT). In
addition, they contain a ribosome binding site between the first
BpiI and BsaI cleavage sites. There are many possible designs for
level 1 destination vectors other than the ones described here.
However, despite these differences, all destination vectors also
have the same general structure in common, namely two convergent
type IIs enzymes flanking the DNA sequence of interest, and
cleavage sites designed to fit the structure of nucleic acids 1 to
7 shown in FIG. 1A.
[0266] FIG. 13 illustrates the cloning of 5 level 1 constructs
(shown in the lower part) from level 0 modules into level 1
destination vectors (shown in the middle part) in a level 1
reaction. Level 1 destination vectors shown in FIG. 12 are used.
Five level 1 constructs, namely a promoter construct, 3 ORF
constructs and a terminator construct are obtained with compatible
cleavage sites for ligation, in the order given, in the subsequent
level 2 reaction.
[0267] FIG. 14 shows the level 2 reaction from the level 1
constructs made as shown in FIG. 13 to produce a functional operon
in a level 2 construct.
[0268] FIG. 15 shows a similar level 2 reaction as shown in FIG.
14, but with an end-linker containing internal BsaI sites for
allowing a further level 2 reaction.
[0269] FIG. 16 illustrates the preparation of 3 additional level 1
constructs (designated 6, 7 and 1, respectively) containing a
second promoter (P2) and two further ORFs (orf4 and orf5). The
level 1 destination vector for orf4 is the last destination vector
of a set of n=7 entry DNAs. The right hand cleavage site thereof
having sequence TGCC allows reuse of the first level 1 destination
vector (pL1P-1 in FIG. 12) for orf5.
[0270] FIG. 17. Cloning of the three additional level 1 constructs
obtained according to FIG. 16 into the level 2i-1 construct made
according to FIG. 15. The reaction product shown at the bottom is a
level 2i-2 construct, since the end-linker used allows a subsequent
level 2 reaction.
[0271] The following figures further illustrate the examples.
[0272] FIG. 18 shows the structure of transcriptional units, level
0 modules, and of destination vectors required for their
cloning.
[0273] (A) The transcriptional units contain up of 5 basic modules
separated by 4 nucleotides sequences that serve as recombination
sites (shown in boxes).
[0274] (B) Level 0 modules shown on the first line are flanked by
BsaI sites. The modules are cloned in a level 0 reaction using the
enzyme BpiI and one of the level 0 destination vectors shown
underneath.
[0275] (C) Strategy for removing internal type IIS recognition
sequences. Removal of a BsaI site in a fragment of interest is done
by amplifying two fragments with primers pr1 and 2 and primers pr3
and 4. Sequences of the BpiI recognition sites in the 5' extensions
in the primers (horizontal arrows) are shown in bold. The two
fragments are cloned using BpiI in the appropriate level 0
destination vector, for example pL0-P in the present example.
[0276] FIG. 19 shows an example for cloning of constructs of level
0, 1 and 2. Antibiotic resistances are indicated.
[0277] (A) Illustrates cloning of level 0 promoter modules.
[0278] (B) Illustrates cloning of a level 1 construct containing a
transcription unit.
[0279] (C) Illustrates cloning of a level 2i-1 construct containing
5 transcription units, TU1 (containing GFP), TU2 (containing p19),
TU3 (containing VP2), TU4 (containing VP5), TU5 (containing VP7)
into destination vector pL2-1.
[0280] (D) Illustrates cloning of a level 2-2 construct containing
11 transcription units. In addition to TU1 to TU5, the construct
contains TU6 (transcription unit with VP3), TU7 (transcription unit
with BAR), TU8 (transcription unit with antibody light chain), TU9
(transcription unit with antibody heavy chain), TU10 (transcription
unit with TMV MP), TU11 (transcription unit with TMV CP).
[0281] FIG. 20 shows in (A) the structure of the 11 level 1
transcription units used in FIG. 19. (B) Shows the structure of
construct pICH51811 that is obtained by cloning of the 11
transcription units cloned in a level 2-2 construct.
[0282] FIG. 21 shows the expression of GFP in a Nicotiana
benthamiana leaf after infiltration of level-2 constructs shown in
FIG. 10. The level-2 constructs were transformed in Agrobacterium
tumefaciens, and the transformed bacteria infiltrated in leaves
using a syringe without a needle. GFP expression was observed at 5
dpi under UV light. The number in parenthesis indicates the number
of transcription units in each infiltrated construct.
[0283] FIG. 22 shows prokaryotic genes cloned as level 0 entry
modules. 3 genes from Pantoea ananatis crtE, I, and B are involved
in lycopene biosynthesis. Other genes known to increase lycopene
expression when overexpressed in E. coli were also cloned: dxs from
Agrobacterium rhizogenes strain K84 and E. coli strain K12, the
ispA genes from the same two species, and the idi and AppY genes
from E. coli strain K12. Two promoters were also cloned as level 0
modules: the Lac Z promoter from pUC19, and a promoter from Pantoea
ananatis.
[0284] FIG. 23 shows in (A) level 1 destination vectors for cloning
of prokaryotic coding sequences. All constructs are in fact
libraries (indicated by the L in the construct name) that have
variable sequences flanking the RBS. (B) shows two sets of end
linkers that contain the LacZ terminator from pUC19.
[0285] FIG. 24 shows a list of level 1 constructs made with the
level 0 modules shown in FIG. 22.
[0286] FIG. 25 shows level 2i-1 constructs. Two libraries were made
with genes for lycopene biosynthesis. Both constructs pICH5648L and
pICH5850L are in fact libraries in which all clones obtained differ
in the sequence flanking the RBS of all three cloned genes.
[0287] FIG. 26 shows addition of two or three more genes to the
operons obtained in FIG. 25. Since a library of different
constructs was placed in the cloning mixes (pICH5447L to pICH5452L
in FIG. 26A, or pICH5455L in FIG. 26B) the constructs obtained
consist of libraries of constructs that differ in the genes present
at positions 5 and 6 or 5, 6 and 7. The number of different gene
combinations for library pICH5920L is in theory 216 possibilities,
without even considering the variation provided by variability in
the 5 RBS regions of the 5 genes per operon.
[0288] FIG. 27 shows that level 2 cloning can be followed by a
level 3. For this, a new type IIs enzyme needs to be used, such as
SapI in the case shown.
[0289] FIG. 28 shows vectors required for level 3 cloning. Level
two destination vectors are flanked by restriction sites of a new
type IIs enzyme (SapI in this example). Level 3 destination vectors
and end-linkers are similar as for level 2, but with the new type
IIS enzyme. Numbers 1 to 8 represent eight different 3 nucleotide
sequences as the cleavage site of SapI.
[0290] FIG. 29A shows a set of destination vectors and end-linkers
for assembly of several level 1 transcription units (or more
generally nucleic acids "na") into a level M destination vector (M
stands for multiplication). Level M destination vectors and
corresponding end-linkers (ELM1 to 7) are designed in such a way
that blocks of assembled transcription units (or nucleic acid
fragments) cloned in level M destination vectors become flanked by
cleavage sites for a type IIs restriction enzyme, which will be
used for the next step of cloning. In contrast to level 2
constructs where types IIs enzymes restriction sites are located at
the end of the constructs in the end-linker, here the type IIs
enzymes flank the assembled nucleic acid fragments, allowing them
to be further subcloned in a new vector.
[0291] FIG. 29B illustrates assembly of three transcription units
(nucleic acid acids na1 to 3) in a level M destination vector. The
linker is chosen from the set of linkers ELM1 to ELM7 such that na3
can be linked to cleavage site S8 of destination vector M.
[0292] FIG. 29C illustrates assembly of two transcription units
(nucleic acids na4 and 5) in a level M destination vector. The
linker is chosen from the set of linkers ELM1 to ELM7 such that na5
can be linked to cleavage site S8 of destination vector M.
[0293] FIG. 29D shows assembly of the two pre-assembled blocks of
transcription units (the reaction products of the reactions shown
in B and C) in a level 2 construct employing a level 2 destination
vector. Details of the restriction sites of destination vectors and
end-linkers are shown in FIGS. 31 and 32 (below).
[0294] FIG. 30 shows that level M can be used as an intermediate
step between level 1 and 2. Examples for color selection,
antibiotic selection and type IIs enzymes for each step are
shown.
[0295] FIG. 31 shows the structure of the entry DNAs as well as
vectors required for level M cloning. A set of n=7 level M
destination vectors and a set of n=7 linkers M is shown. Similarly
as level 2 cloning, level M requires destination vectors and a set
of compatible end-linkers, and allows from 1 to 6 (with sets of n=7
destination vectors and linkers as shown) transcription units (or
nucleic acid fragments) to be assembled in one step. The
orientation and position of type IIS restriction sites in
destination vectors and end-linkers is such that assembled
transcription units (or nucleic acid fragments) in level M
constructs can be excised by a type IIs enzyme, in this case BsaI.
Entry DNAs are flanked by type IIs restriction sites in opposite
orientations (here BpiI sites). The cleavage sites of these two
sites are each identical to one of the seven cleavage sites present
on the left-hand side of level M destination vectors and linkers.
The 7 cleavage sites on the left-hand side of the 7 destination
vectors are unique (and different from one another). The same set
of 7 cleavages sites is present on the left-hand side of the
linkers M.
[0296] FIG. 32 shows and example where blocks of three (FIG. 32a)
and two (FIG. 32b) transcription units are separately pre-assembled
into level M destination vectors before being assembled in a level
2 vector in a subsequent step (FIG. 32c).
[0297] FIG. 33 illustrates a set of n=7 destination vectors and a
set of n=7 end-linkers required for level M (left side of the
figure) and P (right side of the figure). The destination vectors
and end-linkers for level M (left side of the figure) are as shown
in FIG. 31. The structure of entry DNAs for level P cloning is
identical to the structure of entry DNAs for level M, except that
restriction sites for a different type IIs enzymes are present
(here for BsaI). Level M constructs have a structure corresponding
to level P entry DNAs. Thus, level M constructs can be used as
entry DNAs in a level P reaction. Level P constructs become entry
DNAs for a next round of level M cloning.
[0298] FIG. 34a illustrates a level P reaction wherein two level M
constructs are assembled into a level P destination vector using an
end-linker P to give a level P construct.
[0299] FIG. 34b illustrates a level M reaction wherein two level P
constructs are assembled into a destination vector M ("destination
M") using an end-linker M to give a Level M construct.
[0300] FIG. 35: Cloning from level P to level M and vice-versa can
be repeated indefinitely by reusing the same set of vectors and
end-linkers shown in FIG. 33.
[0301] FIG. 36 illustrates the structure of constructs obtained in
successive level M and level P cloning steps. In this example, 90
transcription units (or nucleic acid fragments) are assembled in a
construct (shown at the bottom) in three cloning steps. 5 Sets of 6
transcription units are assembled in parallel in separate reactions
in the first level M cloning step. The resulting 5 constructs are
then assembled in one construct in the following cloning reaction
on level P. This construct and two other compatible constructs
(construction not shown for lack of space), each containing 30
assembled transcription units (or nucleic acid fragments) are then
assembled in a third step of cloning in a level M destination
vector. Boxes with numbers 1 to 7 represent nucleic acid fragments
or transcription units, with such numbers referring only to the
nature of the type IIs enzyme cleavage sites (position as defined
earlier) flanking them, and not to the sequence of the nucleic
acids contained between these two sites.
EXAMPLES
Molecular Biology Reagents
[0302] Restriction enzymes used in this study were purchased from
New England Biolabs and Fermentas. T4 DNA ligase was purchased from
Promega. Plasmid DNA preparations were made by using the NucleoSpin
Plasmid Quick Pure kit (Macherey-Nagel, Duren, Germany) following
the manufacturer protocol. Plasmid DNA concentration was measured
using a Nano Drop.RTM. Spectrophotometer ND-1000 (Peqlab,
Erlangen). The coding DNA for the coat proteins VP2, VP3, VP5 and
VP7 of blue tongue virus serovar 8 was synthesised from Entelechon
GmbH and lack all BpiI, BsaI and Esp3I restriction sites). Level-0
modules were sequenced with primers moclof (SEQ ID NO: 1:
5'-agcgaggaagcggaagagcg) and moclor (SEQ ID NO: 2:
5'-gccacctgacgtctaagaaacc).
Reference Example 1
Standard Cloning Protocol
[0303] A one step-one pot restriction/ligation was setup with
approximately 30 fmol (.about.100 ng for a 5 kb plasmid) of each
fragment (PCR product or plasmid), Promega ligation buffer, 10 U of
the respective restriction enzyme (BsaI, BpiI, or Esp3I), 10 U high
concentrated T4 DNA ligase (Promega), in a 20 .mu.l volume. The
reaction was incubated for 5 hours at 37.degree. C., 5 min
50.degree. C. and 5 min 80.degree. C. The mix was added to 100
.mu.l chemical competent DH10b cells, incubated for 30 min on ice
and transformed by heat shock. Two clones with the expected color
were analysed by restriction analysis and optionally by
sequencing.
Reference Example 2
Cloning of the Canthaxanthin Biosynthesis Operon
[0304] A DNA fragment coding for canthaxanthin biosynthesis was
made by PCR amplification of 4 genes from Pantoea ananatis that are
necessary for biosynthesis of .beta.-carotene (genes crtE, crtY,
crtI and crtB, Ref) and of one gene from Agrobacterium aurantiacum
(crtW) necessary to convert .beta.-carotene to canthaxanthin (ref).
The gene crtW gene is used in addition to the 4 pantoea genes
because the orange/red color of canthaxanthin is easier to see on
agar plates than the yellow color of .beta.-carotene. The Pantoea
ananatis strain was obtained from the DSMZ (cat DSM 30080), and a
fragment containing the crtW gene was synthesised by Mr. Gene GmbH.
An artificial operon containing the genes crtE-W-Y-1-B under
control of the P. ananatis native promoter was made by ligation of
three fragments derived from PCR: fragment 1 containing the
promoter and crtE gene was amplified from P. ananatis genomic DNA
with primers 5'-ttt ggtctc a ggag ggtaccgcacggtctgccaa (SEQ ID NO:
3) and 5'-ttt ggtctc a tcatgcagcatccttaactgacggcag (SEQ ID NO: 4),
fragment 2 containing the crtW gene was amplified from a synthetic
DNA fragment (sequence identical to the native sequence) with
primers 5'-ttt ggtctc a atgagcgcacatgccctgcc (SEQ ID NO: 5) and
5'-ttt ggtctc a tcactcatgcggtgtcccccttggt (SEQ ID NO: 6), and
fragment 3 containing the genes crtY-1-B was amplified from Pantoea
DNA using primers 5'-ttt ggtctc a gtgacttaagtgggagcggctatg (SEQ ID
NO: 7) and 5'-ttt ggtctc a atgtagtcgctctttaacgatgag (SEQ ID NO: 8).
The fragments were assembled by Golden Gate cloning in a target
vector using BsaI. Two BpiI and one Esp3I site present in crtY were
removed using primers containing silent mutations in the
recognition sites.
Reference Example 3
Infiltration Tests
[0305] To check that the constructs are working, at least for one
of the transcriptional units (containing GFP), all level-2
constructs were introduced into Agrobacterium tumefaciens.
Agrobacterium suspensions were infiltrated with a syringe without a
needle into Nicotiana benthamiana leaves. GFP is expressed from all
constructs, as expected from expression cassettes driven by the 35S
promoter. Interestingly, the level of GFP expression was found to
decrease for the largest constructs. This can be explained by the
fact that the GFP gene was always located at the left border in all
constructs; since T-DNA transfer to plant cells occurs from the
right to the left border, and is sometimes incomplete, plant cells
will acquire the GFP cassettes from large constructs less
frequently than from smaller constructs.
Example 1
Generation of the Basic Parts: The Level 0 Modules
[0306] We defined in a first step a generalized eukaryotic
transcriptional unit as the basis for our modular cloning system
(MoClo). This unit was subdivided into five basic modules which
cover the most important features of any transcriptional unit:
promoter (P), 5'UTR (5U), signal peptide (SP), open reading frame
(ORF), and terminator (T, which also includes 3' untranslated
sequences) (FIG. 3). For each module, two unique flanking
recombination sites were defined. We use here the term
`recombination sites` by functional analogy to the recombination
sites used in recombination systems such as the phage P1 Cre-loxP
recombination system. However, for the purpose described here,
`recombination sites` can be any nucleotide sequence of choice of
at least 3 base pairs in length, and will serve as the sequence
where restriction enzyme digestion and ligation will take place; no
recombinase will in fact be used. To guarantee efficient cloning,
these sites were chosen to be non-palindromic and to share a
maximum of two identical consecutive nucleotides. The recombination
site between the promoter fragment and the signal peptide reads
AATG and encodes a start codon preceded by an adenosine, which is
the most common nucleotide in dicotyledonous plants at this
position. The recombination site between the signal peptide and the
rest of the protein (for secreted proteins) was chosen to be AGGT,
with GGT coding for a glycine; this sequence was chosen as the last
aminoacid of the signal peptide can usually be a glycine, and this
allows producing secreted proteins with native sequence. The four
remaining recombination site sequences chosen (GGAG, TACT, GCTT and
CGCT) were selected without any special requirement other than
being unique and non-palindromic, since they are all present in
non-coding DNA.
[0307] The designated DNA fragments are then amplified by PCR with
primers designed to attach the specific recombination site and the
recognition site sequence for the type IIS restriction enzyme BpiI
(FIGS. 5A and 5B). To allow efficient cloning of these PCR
fragments, a set of level 0 destination vectors was constructed
(one vector for each type of level 0 module), each containing two
BpiI restriction sites compatible with the amplified PCR fragments.
Beside the destination plasmids for the five standard elements
(pL0-P, pL0-5, pL0-S, pL0-O and pL0-T) further variants were
included, which allow an easy adaptation of the MoClo system to a
wide variety of projects (FIG. 18). The expression of cytosolic
proteins for example does not require a signal peptide, and these
can be cloned into pL0-SO, spanning the SP and ORF position. All
level 0 destination plasmids are based on a pUC19 backbone, confer
spectinomycin resistance and encode a lacZ.alpha. fragment for
blue/white selection. On both sites of the lacZ.alpha. fragment two
different type IIS recognition sequences--for the enzymes BsaI and
BpiI--are positioned in inverse orientation relative to each other
(FIG. 5A, B). Both recognition sequences are designed to create the
identical four nucleotide recombination site upon digestion (for
example, sequence GGAG on the left side of the destination vector
for cloning of promoter modules). This design allows cloning of the
PCR product(s) for the DNA fragment of interest efficiently via
BpiI--removing the BpiI recognition sites and lacZ.alpha. in the
process--but provides the possibility to release the cloned
fragment with BsaI using the identical recombination sites it was
cloned in.
[0308] The level 0 modules should not contain any of the type IIS
restriction sites used in the MoClo system within the sequence of
the fragments of interest. Beside the already mentioned BsaI and
BpiI, a third type IIs enzyme, Esp3I, is used in the process of
assembly of higher order constructs (see below). Removal of these
sites can be easily done at the time of cloning of level 0 modules
by using primers overlapping the internal BpiI, BsaI or Esp3I
sites, but containing a single silent nucleotide mismatch in the
recognition site. An example for the removal of a single BsaI site
is given in FIG. 18C. The primers pr2 and pr3 introduce a guanine
to cytosine exchange and destroy the BsaI recognition site. The
level 0 module is then assembled from the PCR-fragments (n+1 PCR
fragments when n sites need to be removed) in a one-step one-tube
reaction using BpiI into the appropriate level 0 destination
plasmid. The purification of the PCR reaction prior cloning is
preferable, as primer dimers can be competitors of the PCR product
in the cloning procedure.
[0309] To show the versatility of the system, we cloned a number of
modules for all elements of the transcriptional unit. These include
11 ORFs representing a wide spectrum of biological functions like
immunoglobulins (IgG, heavy and light chain), structural viral
proteins from BTV and PVX (Potato Virus X), the silencing inhibitor
p19, the bar resistance marker and GFP. As an example, we provide
here how a promoter module can be cloned. The 35S promoter fragment
was generated by PCR using 35S promoter specific primers which add
the BpiI recognition sites (underlined) and the promoter module
specific fusion sites (bold). The 35S forward primer comprises:
5'-ttt GAAGACAAGGAG (SEQ ID NO: 9) followed by bases specific for
the 35S promoter, 35S reverse comprises: 5'-ttt GAAGACAAAGTA (SEQ
ID NO: 10) followed by bases specific for the 35S promoter.
[0310] In order to create the level 0 module pICH41373 (pL0-P with
the 35S promoter) by a BpiI dependent Golden gate cloning reaction,
the following reaction mix was added into a single tube (FIG.
19A):
TABLE-US-00003 2 .mu.l 10 .times. T4-ligase buffer (Promega) 1
.mu.l high concentrated T4-ligase (Promega; 10 U/.mu.l) 1 .mu.l
Bpil restriction enzyme (Fermentas 10 U/.mu.l) 1 .mu.l 30 fmol
pL0-P destination plasmid 1 .mu.l specific PCR product
(column-purified to eliminate primer dimers) of the 35S promoter
(generated by standard PCR with primers describe above) 14 .mu.l
water 20 .mu.l total
[0311] The reaction was incubated for 5 hours at 37.degree. C., and
10 min 80.degree. C. The mix was added to 100 .mu.l chemical
competent DH10b cells, incubated for 30 min on ice and transformed
by heat shock. After plating on LB agar plates containing
spectinomycin (100 .mu.g/ml) and growing over night at 37.degree.
C., two white clones were analyzed by restriction analysis and by
sequencing.
[0312] In contrast to the number of ORFs, the number of commonly
used promoters and terminator sequences available for expression of
heterologous proteins in plants is much lower. To avoid repetitive
sequences in planned multigene constructs, we therefore cloned
several Arabidopsis thaliana promoter and terminator sequences from
genes which show a high basic expression level. After sequencing,
the level 0 modules form the bottom level in the hierarchal MoClo
system. A summary of all level 0 modules used in this study is
presented in the table below:
TABLE-US-00004 Module type and construct Reference or accession
number Relevant characteristics number Promoter (P) pICH41373 35S
promoter CaMV.sup.1 pICH41551 ST-LS1 (Stem and leaf specific)
promoter S. tuberosum;.sup.2 pICH42755 34S promoter FMV.sup.3
pICH42760 Spm promoter Zea mays.sup.4 pICH44157 RBCS (RuBisCO Small
subunit 1b) promoter A. thaliana; At5g38430; This work pICH45131
LHB1B2 promoter A. thaliana; At2g34420; This work pICH45145 LHCB5
promoter A. thaliana; At4g10340; This work pICH45167 RRM-containing
protein promoter A. thaliana; At1g70200; This work pICH50581 ACT2
(Actin 2) promoter A. thaliana; At3g18780; This work 5'UTR (U)
pICH46501 Tabacco mosaic virus .quadrature.fragment This work ORF
pICH41531 sGFP codon optimized .sup.5 pICH42222 Basta .TM.
resistance protein S. hygroscopicus.sup.6 (Phosphinothricin
acetyltransferase) pICH44022 P19 Tomato bushy stunt virus silencing
inhibitor .sup.7 pICH45502 BTV (blue tongue virus) VP2 This work
pICH45512 BTV (blue tongue virus) VP3 This work pICH45526 BTV (blue
tongue virus) VP5 This work pICH45531 BTV (blue tongue virus) VP7
This work pICH48348 PVX CP This work pICH48367 PVX 25K MP This work
pICH49488 IgG.sub.1 Light chain with native signal peptide This
work pICH49500 IgG.sub.1 Heavy chain with native signal peptide
This work Terminator (T) pICH41432 Ocs terminator A.
tumefaciens.sup.8 pICH41414 35S terminator CaMV pICH44300 ACT2
(Actin 2) terminator A. thaliana; At3g18780; This work pICH44311
TGG1 (Thioglucoside Glucohydrolase 1) A. thaliana; At5g26000; This
terminator work pICH44344 GCRP (glycine-rich protein) terminator A.
thaliana; At1g67870; This work pICH44355 AAC1 (ADP/ATP carrier
protein 1) terminator A. thaliana; At3g08580; This work pICH44377
PRXR1 (Peroxidase 1) terminator A. thaliana; At4g21960; This work
pICH44388 AGP18 (Arabinogalactan protein 18) terminator A.
thaliana; At4g37450; This work pICH44393 GAE6 (UDP-D-Glucoronate
4-Epimerase 6) A. thaliana; At3g23820; This terminator work
pICH49344 Nos terminator A. tumefaciens.sup.9 .sup.1Guilley, H.,
Dudley, R. K., Jonard, G., Balazs, E. & Richards, K. E.
Transcription of Cauliflower mosaic virus DNA: detection of
promoter sequences, and characterization of transcripts. Cell 30,
763-773 (1982). .sup.2Stockhaus, J., Eckes, P., Blau, A., Schell,
J. & Willmitzer, L. Organ-specific and dosage-dependent
expression of a leaf/stem specific gene from potato after tagging
and transfer into potato and tobacco plants. Nucleic Acids Res 15,
3479-3491 (1987). .sup.3Sanger, M., Daubert, S. & Goodman, R.
M. Characteristics of a strong promoter from figwort mosaic virus:
comparison with the analogous 35S promoter from cauliflower mosaic
virus and the regulated mannopine synthase promoter. Plant Mol Biol
14, 433-443 (1990). .sup.4Raina, R., Cook, D. & Fedoroff, N.
Maize Spm transposable element has an enhancer-insensitive
promoter. Proc Natl Acad Sci USA 90, 6355-6359 (1993). .sup.5Chiu,
W. et al. Engineered GFP as a vital reporter in plants. Curr Biol
6, 325-330 (1996). .sup.6Thompson, C. J. et al. Characterization of
the herbicide-resistance gene bar from Streptomyces hygroscopicus.
Embo J 6, 2519-2523 (1987). .sup.7Marillonnet, S., Thoeringer, C.,
Kandzia, R., Klimyuk, V. & Gleba, Y. Systemic Agrobacterium
tumefaciens-mediated transfection of viral replicons for efficient
transient expression in plants. Nat Biotechnol 23, 718-723 (2005).
.sup.8De Greve, H. et al. Nucleotide sequence and transcript map of
the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase
gene. J Mol Appl Genet 1, 499-511 (1982). .sup.9Depicker, A.,
Stachel, S., Dhaese, P., Zambryski, P. & Goodman, H. M.
Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl
Genet 1, 561-573 (1982).
Example 2
Assembly of Transcriptional Units: The Level 1 Constructs
[0313] The next level of cloning consists of assembling several
level 0 modules into a complete transcriptional unit in a level 1
reaction. Since assembly is performed by Golden Gate cloning using
the enzyme BsaI, no BsaI restriction site is left in the resulting
level 1 construct. Therefore, to be able to later subclone the
assembled transcriptional unit into higher level constructs, two
restriction sites of a second type IIS restriction enzyme also have
to be present flanking the assembled fragment. We therefore created
level 1 destination vectors containing two BpiI restriction sites
flanking the lacZ.alpha. fragment, in addition to the two BsaI
restriction sites needed for cloning of the transcription unit
(pL1F-1 to pL1F-7, FIG. 4). If, as for level 0 destination vectors,
the cleavage sites of the two different type IIS enzymes (BsaI and
BpiI) overlapped, all level 1 transcriptional unit fragments would
be flanked by the same GGAG and CGCT recombination sites (which
originate from the promoter and terminator modules, respectively).
The presence of identical overhangs flanking each transcriptional
unit would prohibit the creation of higher level multigene
constructs with a defined linear order. Consequently a series of
seven level 1 destination vectors was designed in which the BpiI
cleavage sites generate two recombination sites with new
specificities for each vector (for example sites TGCC and GCAA for
level 1 vector position 1, FIGS. 4 and 5B). These sites are
compatible from one vector to the next so that assembled
transcriptional units can be subcloned in a one-pot reaction from
the level 1 constructs into a level 2 construct. If the sites of
all level 1 constructs were defined strictly for linear assembly,
the number of level 1 destination vectors would need to be as high
as the number of transcriptional units that an experimenter would
wish to assemble in a final construct. However, to avoid the
construction and consideration of too many level 1 destination
vectors, the spatial order of overhangs was designed to be circular
instead of linear, as the first recombination site at position 1
(TGCC) is also the final site at position 7. So, a level 1
construct for position 1 can be reused later at a virtual position
"8" (FIG. 4). Therefore, such a set of 7 level 1 destination
vectors will allow an unlimited number (regarding the design, not
the physical size of DNA) of transcriptional units to be cloned in
a final nucleic acid construct of interest, although by incremental
steps of up to 6 constructs at a time (see further description
below for such assembly).
[0314] Between the upstream BpiI and BsaI sites, we also introduced
additional restriction sites for analytical restriction digests: an
EcoRI site is present in each level 1 destination plasmid, whereas
a second restriction site is specific for each position (FIG. 5B).
Beside the seven level 1 destination vectors for the assembly of
transcriptional units in the forward direction, a set of level 1
destination vectors for cloning of transcription units in the
opposite orientation was also created. Here the BsaI overhangs are
in reverse complement orientation, but accept the same level 0
modules as for the other orientation (pL1R-1 to pL1-R7, FIG.
4).
[0315] Since our transient plant expression system is based on
Agrobacterium tumefaciens, all plasmids have a broad host range RK2
origin of DNA-replication and left border (LB) and right border
(RB) T-DNA sequences to allow T-DNA transfer into the plant cell.
These two features allow testing the functionality of level 1
constructs by plant infiltration. It is also possible to make
similar vectors for allowing expression in other eukaryotic hosts
such as yeast, insect or mammalian cells or in prokaryotes. The
level 1 destination vectors encode an ampicillin resistance gene
and a lacZ.quadrature. fragment flanked by BpiI and BsaI sites.
[0316] To test the efficiency of the assembly of level 0 modules
into level 1 transcriptional units (level 1 constructs), 11
artificial transcriptional units were designed (promoters and
terminators were randomly assigned to ORFs without consideration
for level of expression since all constructs in this study were
made purely as an exercise to demonstrate the ability of the MoClo
system for cloning of multigene constructs), and were (again
randomly) assigned to one of the seven level 1 positions. In 11
independent cloning reactions, the level 0 modules were combined
with the respective level 1 destination vectors, T4-DNA ligase and
the restriction enzyme BsaI in a one-tube one-step reaction (FIG.
20). The different antibiotic resistances of level 0 and level 1
destination plasmids in combination with the blue/white selection
represent a very convenient way to screen for correctly assembled
level 1 constructs. After transformation, the reactions were spread
on ampicillin and X-Gal containing plates and the numbers of white
and blue clones were counted. The number of white, and therefore
correct, colonies varied from approximately 16000 to 180000,
whereas only a minor fraction (<1%) of blue colonies was present
in two out of eleven reactions (FIG. 10). Two white colonies from
each reaction were analyzed by an analytical restriction digest
with BpiI (which cleaves on both sides of the assembled
transcriptional unit). All 22 tested plasmids contained a fragment
of the expected size.
[0317] We provide here as an example how the cloning reaction was
set up for one of the transcription units. In order to create the
level 1 construct pICH50711 by a BsaI dependent Golden gate cloning
reaction, the following reaction mix was added into a single tube
(FIG. 19 B):
TABLE-US-00005 2 .mu.l 10 .times. T4-ligase buffer (Promega) 1
.mu.l high concentrated T4-ligase (Promega; 10 U/.mu.l) 1 .mu.l
Bsal restriction enzyme (NEB; 10 U/.mu.l) 1 .mu.l 30 fmol pL1F-1
destination plasmid 1 .mu.l 30 fmol pICH41373 (level 0 promoter
module, 35S promoter) 1 .mu.l 30 fmol pICH46501 (level 0 5'UTR
module, TMV untraslated region) 1 .mu.l 30 fmol pICH41531 (level 0
ORF module, GFP) 1 .mu.l 30 fmol pICH41432 (level 0 terminator
module, Ocs terminator) 11 .mu.l water 20 .mu.l total
[0318] The reaction was incubated for 5 hours at 37.degree. C., 5
min 50.degree. C. and 5 min 80.degree. C. The mix was added to 100
.mu.l chemical competent DH10b cells, incubated for 30 min on ice
and transformed by heat shock. After plating on LB agar plates
containing ampicillin (100 .mu.g/ml) and growing over night at
37.degree. C., two white clones were analyzed by restriction
analysis and optionally by sequencing.
Example 3
Design of Multigene Constructs: The Level 2
[0319] As all other MoClo constructs, multigene level 2 constructs
are assembled from lower level (here level 1) constructs using a
one-pot restriction-ligation. In this case, assembly is performed
using the enzyme BpiI. Level 2 destination vectors carry a
kanamycin resistance gene, in accordance with the principle that a
specific selection marker is assigned to each level of cloning,
allowing effective counter-selection against entry plasmid
backbones. Level 2 destination vector backbones do not contain any
type IIs restriction sites used in the MClo system, other that the
recognition sites used for the cloning of the inserts. In contrast
to level 0 and level 1, the visible selectable marker used for
level 2 constructs is not a lacZ gene, but an artificial bacterial
operon containing 5 genes (see Reference Example 2) that lead to
biosynthesis of canthaxanthin, a red (more precisely salmon/orange)
colored carotenoid pigment. A lacZ gene for blue-white selection
would have also worked for this step, but the choice of a new color
selectable marker is explained below in the paragraph on level 2i.
The cantaxanthin operon in level 2 destination vector pL2-1 is
flanked by two BpiI sites that create TGCC and GGGA overhangs after
digestion (FIG. 4). The TGCC overhang is compatible with the level
1 construct for position 1, while the GGGA represents an overhang
which is unique to level 2 destination plasmids. Depending on the
number of level 1 constructs that are subcloned in one step (from
one to a maximum of 6 transcription units), the last overhang of
the assembled DNA fragment will be different for each of the six
possibilities. To connect this end with the GGGA overhang of the
level 2 destination vector, a set of seven end-linkers (pELE-n) was
designed (FIG. 4, the seventh linker is necessary only when using
the other level 2 destination vectors described below). Like the
level 1 modules, the end-linker plasmids confer ampicillin
resistance, and are themselves flanked by BpiI sites. These two
features make them compatible with standard level 1 destination
vectors (and constructs obtained therefrom) in a BpiI-based level 2
Golden Gate cloning reaction. End-linkers can consist of any DNA
sequence, and serve the purpose of joining two chosen type IIs
enzyme cleavage sites. For example, the sequence of linker pELE-1
is (SEQ ID NO: 11) gaagac aa GCAA gaggatgcacatgtgaccga GGGA tt
gtcttc (bold is the Bpi recognition sites, underlined are the
cleavage sites, and in between is the joining linker sequence). The
end linkers are cloned in a pUC19 based plasmid (but could also be
cloned in other backbones).
[0320] At first glance the level 1 constructs designed for a
defined position cannot be reused in a different context. For
example, a level 1 construct made for subcloning at position three
cannot be used without two other constructs for position 1 and 2.
Placing the same transcriptional unit at position 1 could be done
by recloning the same level 0 modules in a level 1 destination
vector specific for position 1. However, a possibility to reduce
the need for extensive recloning of the same construct for
different positions is given by the periodical design of the level
1 overhangs. Here the relative position of, for example a level 1
position 3 construct, can easily be shifted to the relative first
position, when the left overhang from the level 2 destination
vector would read ACTA instead of TGCC. Here the first two
positions would be virtually deleted, shifting position 3 to a
relative position 1. A set of seven level 2 destination vectors
created for this purpose is shown in FIG. 4 (pL2-1 to pL2-7).
Together with the seven end-linker plasmids, these 14 plasmids
allow to realize every overhang combination in a level 2
destination vector. The identical flexibility only based on level 2
destination vectors without the end-linkers would require 36
different plasmids.
[0321] To test cloning of several level 1 transcriptional units
into level 2 constructs, 5 different restriction-ligation reactions
were set up to clone from 2 up to 6 transcriptional units in a
single step. The restriction-ligation reactions each contains from
2 to 6 level 1 transcriptional unit constructs (pICH50711,
pICH50721, pICH49722, pICH49733, pICH50731, pICH50741 FIG. 10), one
appropriate end-linker (pELE-2 to pELE-6), and the destination
vector pL2-1.
[0322] The reaction was incubated for 5 hours at 37.degree. C. and
10 min 80.degree. C. The mix was added to 100 .mu.l chemical
competent DH10b cells, incubated for 30 min on ice and transformed
by heat shock. The transformation was plated on LB agar plates
containing kanamycin (100 .mu.g/ml) and the plate incubated over
night at 37.degree. C.
[0323] Considering all level 1 cloning experiments, the number of
white colonies obtained per transformation, which gives a measure
of the cloning efficiency, decreased from approximately 33000 (for
two level 1 modules plus end linker) to 150 (six modules plus
end-linker), and the percentage of red colonies raised from 0.02%
to 10% (FIG. 10). Six white colonies were tested from each level 2
assembly by an analytical endonuclease restriction digest and all
were found to be correct.
Example 4
The Level 2i-1
[0324] As we have shown above, the assembly of up to six
transcription units to produce a 24 kb construct (pICH51201) can be
done in a one-step and one-tube level 2 reaction. However the final
level 2 constructs are in a "closed" status and no additional genes
can be inserted since no type IIS restriction sites are left in the
construct. An entry option can be provided when modified
end-linkers containing additional type IIS restriction sites
(pELB-n) are used in the assembly of the level 2 constructs (FIG.
4). This type of construct was named level 2i since it is based on
level 2 backbones but represents also an intermediate step to
extend the number of genes in the construct beyond six. This
intermediate construct can be used both as a destination vector for
a final construct (level 2-2) or once again to construct the next
level destination vector (level 2i-2), in case more than 6 more
genes still need to be added to the construct. Given these
alternative possibilities, the same visual selectable marker (such
as LacZ.alpha. for blue/white selection) cannot be used for all
possible cloning steps. It is for this reason that we developed a
second color selectable marker, that we developed based on
carotenoid biosynthetic genes. With such marker, every cloning step
can be performed using color selection, for example from red to
white for level 2, red to blue for level 2i, blue to white for
level 2-2, blue to red for level 2i-2, etc. . . .
[0325] As an example of end-linker sequence, we provide the
sequence features of plasmid pELB-1. pELB-1 contains the sequence
(SEQ ID NO: 12) gaagac aa tgcc t gagacc (bold BpiI recognition
site, underlined cleavage site of BpiI and BsaI, italics BsaI
recognition site) followed by a puc19 fragment containing the LacZ
alpha fragment
(gcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctca-
ctcattaggcac
cccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacagg-
aaacagctatgaccat
gattacgccaagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattcactggcc-
gtcgttttacaacg
tcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgt-
aatagcgaagagg
cccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtalittctcct-
tacgcatctgtgcgg
tatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacac-
ccgccaacacccgc
tgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgac (SEQ ID
NO: 13), followed by sequence ggtctc a ggga tt gtcttca (SEQ ID NO:
14) (as before, bold BpiI recognition site, underlined cleavage
site of BpiI and BsaI, italics BsaI recognition site). The sequence
of the end-linker shown above is cloned in a pUC19-based vector
(that does not contain additional LacZ fragment sequences), but can
also be cloned in other plasmid backbones).
[0326] To test cloning of level 2i constructs, two
restriction-ligations were set up as for level 2, except that the
end-linker was replaced by an end-linker containing BsaI sites and
a lacZ gene (constructs pICH51212 and pICH51226, FIG. 10). We
provide here in more detail the set up for cloning of pICH51212 as
an example. In order to create the level 2i construct pICH51212 by
a BpiI dependent Golden gate cloning reaction, the following
reaction mix was added into a single tube (FIG. 19 C):
TABLE-US-00006 2 .mu.l 10 .times. T4-ligase buffer (Promega) 1
.mu.l high concentrated T4-ligase (Promega; 10 U/.mu.l) 1 .mu.l
Bpil restriction enzyme (Fermentas 10 U/.mu.l) 1 .mu.l 30 fmol
pL2-1 (destination vector) 1 .mu.l 30 fmol pICH50711 (level 1
construct for position 1, transcription unit with GFP) 1 .mu.l 30
fmol pICH50721 (level 1 construct for position 2, transcription
unit with p19) 1 .mu.l 30 fmol pICH49722 (level 1 construct for
position 3, transcription unit with VP2) 1 .mu.l 30 fmol pICH49733
(level 1 construct for position 4, transcription unit with VP5) 1
.mu.l 30 fmol pICH50731 (level 1 construct for position 5,
transcription unit with VP7) 1 .mu.l 30 fmol pELB-5 (end-linker for
position 6) 9 .mu.l water 20 .mu.l total
[0327] In contrast to previous constructs, red/blue selection is
performed rather than red/white selection. In addition to red and
blue colonies, a few colonies had a dark green color. These contain
incorrect plasmids that have both the canthaxanthin operon and a
lacZ gene. The number of colonies containing correctly assembled
plasmids (blue colonies), and the ratio of red to blue colonies for
pICH51212 and pICH51226 were comparable with the level 2 construct
made from a similar number of entry clones, pICH51191 and
pICH51201.
Example 5
The Level 2-2
[0328] As a starting point for the introduction of up to six
further level 1 constructs, the level 2i-1 construct pICH51212 was
chosen as a destination vector. This construct contains, beside
five level 1 modules, a lacZ.alpha. end-linker providing two BsaI
restriction sites. In contrast to the previously described cloning
of level 1, level 2 or level 2i constructs, which required either
BsaI (level 1) or BpiI (level 2 and level 2i) alone, we have to use
here both enzymes at the same time. BsaI allows reopening the level
2i backbone and provides defined overhangs which are compatible
with the level 1 modules released by BpiI. Since two type IIS
restriction enzymes have to be used at the same time and the target
plasmid has already a size of 22 kb, we again tested the efficiency
of the Golden Gate cloning for the introduction of either one to up
to six level 1 modules simultaneously.
[0329] The results for the construction of plasmids pICH51761 to
pICH51811 show that the cloning efficiency decreases dependent on
the number of incorporated transcription unit fragment constructs
(FIG. 10). Interestingly, the rate with which the cloning
efficiency drops is similar to the earlier analyzed set of level
2-1 plasmids (pICH51161 to pICH51201) despite the different target
plasmid size of 22 kb versus 4 kb. Since no positive clone was
obtained for the largest construct, we repeated the cloning using
different reaction conditions (FIG. 10, for pICH1811*).
[0330] The following reaction mix was set up (FIG. 19D):
TABLE-US-00007 2 .mu.l 10 .times. T4-ligase buffer (Promega) 1
.mu.l high concentrated T4-ligase (Promega; 10 U/.mu.l) 0.5 .mu.l
Bpil restriction enzyme (Fermentas 10 U/.mu.l) 0.5 .mu.l Bsal
restriction enzyme (NEB 10 U/.mu.l) 4.67 .mu.l 40 fmol pICH51212
level 2i-1 construct 0.72 .mu.l 40 fmol pICH50741 (level 1
construct for position 1) 0.75 .mu.l 40 fmol pICH50751 (level 1
construct for position 2) 0.69 .mu.l 40 fmol pICH50761 (level 1
construct for position 3) 0.75 .mu.l 40 fmol pICH50771 (level 1
construct for position 4) 0.58 .mu.l 40 fmol pICH50781 (level 1
construct for position 5) 1.17 .mu.l 40 fmol pICH50791 (level 1
construct for position 6) 0.70 .mu.l 40 fmol pELE-4 (end linker)
5.97 .mu.l water 20 .mu.l total
[0331] The mix was incubated in a thermocycler with the following
parameters: incubation for 2 minutes at 37.degree. C., 5 minutes at
16.degree. C., both steps repeated 45 times, followed by incubation
for 5 minutes at 80.degree. C. and 10 minutes at 80.degree. C. The
reaction mix was transformed in E. coli chemically competent cells,
and an aliquot of the transformation plated on a LB plate
containing Kanamycing and X-gal. These parameters greatly increased
cloning efficiency since 2685 white colonies were obtained (for the
whole transformation) and no blue colony (FIG. 10). Plasmid DNA
from 6 randomly picked colonies was analyzed. All were found to
contain the expected correct construct.
[0332] We have therefore shown here that complex constructs
containing many transcription units (eleven as shown here,
consisting of 44 individual basic modules) can easily be assembled
by a series of three easy-to-perform one-pot reactions, and with
extremely high cloning efficiency. The largest construct made in
this study (pICH51811) has a size of 34 kb. Considering the
relative efficiency with which this construct and its precursors
were obtained, it is likely that we have not yet reached the upper
size limit for constructs that can be made using this technology.
To make larger constructs starting from those that we have
described here, the next step would be to remake the final
construct (pICH51811), but using an end-linker that would add two
restriction sites for the enzyme Esp3I (end-linker pELR-4, FIG. 4),
and use the resulting plasmid as a destination vector to add one or
several additional genes.
Example 6
Infiltration Tests
[0333] To check the constructs, at least for one of the
transcriptional units (containing GFP), all level 2 constructs were
introduced into Agrobacterium tumefaciens. Agrobacterium
suspensions were infiltrated with a syringe without a needle into
Nicotiana benthamiana leaves. GFP is expressed from all constructs
(FIG. 21), as expected from expression cassettes driven by the 35S
promoter. Interestingly, the level of GFP expression was found to
decrease for the largest constructs. This can be explained by the
fact that the GFP gene was always located at the left border in all
constructs; since T-DNA transfer to plant cells occurs from the
right to the left border, and is sometimes incomplete, plant cells
will acquire the GFP cassettes from large constructs less
frequently than from smaller constructs.
Example 7
Cloning of Bacterial Operons
Level 0 Modules:
[0334] As an example for cloning of bacterial operons, we chose to
work with a carotenoid biosynthesis pathway since carotenoids are
easily visible in the colonies, for example as a red color for
lycopene. We chose the Pantoea ananatis Zeaxanthin biosynthesis
pathway, since all genes of the pathway are known and sequenced
(Misawa et al., Journal of Bacteriology, 1990, 172:6704-6712).
Three genes of this pathway are required for lycopene biosynthesis
crtE, crtI and crtB. We PCR-amplified all three genes and cloned
them in vector pLO-SO (FIG. 18) as described for eukaryotic coding
sequences. The resulting level 0 constructs are shown in FIG. 22.
We also cloned several genes known to increase synthesis of
lycopene when over-expressed in E. coli cells expressing the three
crt genes necessary for lycopene production. These genes are dxs,
ispA, idi and AppY. The genes for dxs and ispA were amplified and
cloned from both E. coli strain K12 and from Agrobacterium
rhizogenes strain K84. The genes for idi and AppY were cloned only
from E. coli strain K12. In addition, the LacZ promoter present in
pUC19 and the pantoea ananatis promoter from the Zeaxanthin
biosynthesis operon were also cloned as level 0 modules (FIG.
22).
Level 1 Destination Vectors for Cloning of Coding Sequences:
[0335] Level 1 destination vectors for cloning of bacterial coding
sequences are different from destination vectors for cloning
eukaryotic transcription units, since they are designed for cloning
of individual coding sequences rather than complete transcription
units. Moreover, they also provide a bacterial ribosome binding
site (RBS) to the cloned coding sequence. Instead of making vectors
with a defined RBS sequence, we made vectors with a degenerate RBS
to provide a range of expression levels (FIG. 23A). For example,
vector pICH4862L was made by PCR amplification of the LacZ fragment
from pUC19 using primers prok1-1deg (SEQ ID NO: 15)
(tttcgtctcattcagaagacat TGCC nv agga dnnnnnn AATG
ggagaccttatgaccatgattacgccaagc, in bold is the core sequence of the
RBS and flanking it is degenerate sequence, the underlined sequence
is the cleavage sites of the BpiI or BsaI restriction sites) and
prok1-2 (SEQ ID NO: 16) (tttcgtctcacttagaagacaa TTGC AAGC
tgagaccttatgcggcatcagagcagattgt). In the above sequence, v stands
for a, c or g; d stands for a, t or g; and n stands for a, t, g or
c. This PCR product was cloned using DraIII in a pUC19-based
plasmid backbone containing compatible DraIII sites. Single
colonies were not picked since the result of this cloning is a
library. Instead, the entire library was grown in E. coli and
plasmid DNA prepared from the library. A similar procedure was
repeated for the other 6 destination vectors, but using different
primers for the different type IIs cleavage sites.
End Linkers:
[0336] Since for this experiment we are cloning carotenoid genes,
end-linkers and level 2 destination vectors were made that do not
already contain carotenoid genes. End linkers in particular were
made in which the sequence of the linker consists of the Lac Z
terminator sequences from plasmid pUC19. The two sets of end
linkers are shown in FIG. 23B.
Level 1 Constructs:
[0337] A set of level 1 constructs was constructed from level 0
modules. Promoter modules were cloned in vector pL1F-1 (FIG. 12)
and the coding sequences in vectors pICH5025L to pICH2030L for the
different positions expected in the final construct (FIG. 24).
Cloning was performed as a one-tube cloning reaction using the
selected level 0 module and the chosen destination vector (or
vector library) using the enzyme BsaI as described for cloning of
level 1 eukaryotic transcription units. The 6 genes cloned as level
0 modules and known to increase lycopene production in E. coli were
all cloned for positions 5, 6 and 7.
Level 2i-1 Constructs:
[0338] Two constructs were made containing the lycopene
biosynthesis genes crtB, crtE and crtI and either the lac Z
promoter or the Pantoea ananatis promoter. No terminator cloned as
level 1 module was used since the terminator is provided here by
the end-linker (FIG. 25). The resulting constructs, pICH5848L and
pICH5850L are in fact two different libraries, in which all
constructs have different RBS sequences for each of the three
genes. The colonies displayed a wide variation in the amount of red
color, as expected from such libraries. The libraries were grown
and plasmid DNA prepared from them.
Level 2i-2 Constructs:
[0339] The next step consists of adding two or three genes to the
previous constructs to try to increase the amount of lycopene
produced. Since we have 6 genes available cloned at three different
positions, there are too many possible combinations to test them
all individually. Instead, libraries can be made in which any of
the 6 genes will be cloned randomly at any of the two or three
positions (position 5 and 6, or 5, 6 and 7, FIGS. 26 A and B). Four
different libraries were made by setting up 4 reactions by adding
into one tube all the constructs depicted in FIG. 26A (two
reactions with either the LacZ promoter or the Pantoea promoter) or
26B (two reactions as well) and incubating the mixes in presence of
BsaI and BpiI enzymes and ligase (as described previously for level
2i-2 for eukaryotic constructs). Colonies with a wide range of
intensity for the red color were obtained (FIG. 26B), with many
colonies having a stronger red that the parental 2i-1 constructs.
To test that genes (from the 6 selected genes) had been cloned
randomly at the three positions, 8 constructs from intense red
colonies were sequenced. All constructs contained a different
combination of genes. All sequenced constructs contained a dxs gene
at least at one of the three positions; this is not surprising,
since dxs is known to be the gene with the strongest effect on
lycopene production among all enhancer genes tested as reported in
the literature.
Sequence CWU 1
1
16120DNAArtificial SequencePCR primer 1agcgaggaag cggaagagcg
20222DNAArtificial SequencePCR primer 2gccacctgac gtctaagaaa cc
22334DNAArtificial SequencePCR primer 3tttggtctca ggagggtacc
gcacggtctg ccaa 34437DNAArtificial SequencePCR primer 4tttggtctca
tcatgcagca tccttaactg acggcag 37530DNAArtificial SequencePCR primer
5tttggtctca atgagcgcac atgccctgcc 30635DNAArtificial SequencePCR
primer 6tttggtctca tcactcatgc ggtgtccccc ttggt 35734DNAArtificial
SequencePCR primer 7tttggtctca gtgacttaag tgggagcggc tatg
34834DNAArtificial SequencePCR primer 8tttggtctca atgtagtcgc
tctttaacga tgag 34915DNAArtificial Sequencepart of forward primer
9tttgaagaca aggag 151015DNAArtificial Sequencepart of reverse
primer 10tttgaagaca aagta 151144DNAArtificial Sequencelinker pELE-1
11gaagacaagc aagaggatgc acatgtgacc gagggattgt cttc
441219DNAArtificial SequencepELB-1 12gaagacaatg cctgagacc
1913578DNAArtificial SequenceLacZ alpha fragment 13gcagctggca
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 60tgagttagct
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
120tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
catgattacg 180ccaagcttgc atgcctgcag gtcgactcta gaggatcccc
gggtaccgag ctcgaattca 240ctggccgtcg ttttacaacg tcgtgactgg
gaaaaccctg gcgttaccca acttaatcgc 300cttgcagcac atcccccttt
cgccagctgg cgtaatagcg aagaggcccg caccgatcgc 360ccttcccaac
agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt
420acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat
ctgctctgat 480gccgcatagt taagccagcc ccgacacccg ccaacacccg
ctgacgcgcc ctgacgggct 540tgtctgctcc cggcatccgc ttacagacaa gctgtgac
5781420DNAArtificial Sequencepart of pELB-1 14ggtctcaggg attgtcttca
201573DNAArtificial SequencePCR primer 15tttcgtctca ttcagaagac
attgccnvag gadnnnnnna atgggagacc ttatgaccat 60gattacgcca agc
731661DNAArtificial SequencePCR primer 16tttcgtctca cttagaagac
aattgcaagc tgagacctta tgcggcatca gagcagattg 60t 61
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