U.S. patent application number 11/546609 was filed with the patent office on 2007-10-18 for modular genomes for synthetic biology and metabolic engineering.
Invention is credited to Robert A. Holt.
Application Number | 20070243617 11/546609 |
Document ID | / |
Family ID | 38309568 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070243617 |
Kind Code |
A1 |
Holt; Robert A. |
October 18, 2007 |
Modular genomes for synthetic biology and metabolic engineering
Abstract
The invention provides methods and compositions for assembling a
modular replacement genome in a host microorganism. After such
assembly, the host organism's genome is inactivated or ablated to
permit full control of host cellular functions by the replacement
genome. A modular replacement genome comprises an assembly of
nucleic acid fragments, or segments, derived from one or more
natural organisms or from synthetic polynucleotides or from a
combination of both. Such an assembly, or set, of segments making
up a replacement genome comprises a substantially complete set of
genes and regulatory elements for carrying out minimal life
functions under predefined culture conditions. The invention
provides modular genomes having modules that are amenable to facile
replacement, deletion, and/or additions. Such modules may be
synthetic polynucleotides and may be designed for controlling gene
content, excluding of genes that encode inhibitors or otherwise
undesirable competing enzymes that divert a host cell from desired
metabolic/synthetic processes; modifying codon usage to maximize or
minimize protein production; modifying regulatory elements,
including promoters, enhancers, repressors, activator, or the like,
to modulate gene expression; balancing enzymatic and transport
activities to optimize fluxes of substrates, intermediates, and
products in metabolic pathways, and like objectives.
Inventors: |
Holt; Robert A.; (North
Vancouver, CA) |
Correspondence
Address: |
Stephen C. Macevicz;Technology Development Office
BC CANCER AGENCY
675 West 10th Avenue
Vancouver
BC
V5Z 1L3
CA
|
Family ID: |
38309568 |
Appl. No.: |
11/546609 |
Filed: |
October 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60725630 |
Oct 13, 2005 |
|
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Current U.S.
Class: |
435/471 ;
435/252.3; 435/320.1; 536/23.7 |
Current CPC
Class: |
C12N 15/70 20130101;
C12N 15/90 20130101; C12N 2800/30 20130101 |
Class at
Publication: |
435/471 ;
435/252.3; 435/320.1; 536/023.7 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C07H 21/04 20060101 C07H021/04; C12N 15/00 20060101
C12N015/00; C12N 15/74 20060101 C12N015/74 |
Claims
1. A method of assembling a replacement genome in a host organism
having a host genome, the method comprising the steps of: (a)
providing a plurality of segments that cover a replacement genome,
each segment being associated with one or more recombination
elements; (b) transforming or co-transforming the host organism
with one or more segments to form a precursor genome, the precursor
genome being a recombinant of the one or more segments or a
recombinant of a prior precursor genome and the one or more
segments, such recombinant being formed by recombination of the
recombination elements associated with the one or more segments;
(c) repeating step (b) with segments of a predetermined ordering
until the replacement genome is formed; and (d) removing the host
genome.
2. The method of claim 1 wherein said step of transforming or
co-transforming comprises co-transforming said host organism with
said plurality of said segments so that said replacement genome is
formed.
3. The method of claim 2 wherein each end of each of said segments
of said plurality has a unique overlapping sequence region with an
end of another said segment of said plurality to establish said
predetermined ordering upon formation of said replacement
genome.
4. The method of claim 1 wherein said step of transforming or
co-transforming comprises transforming the host organism with a
segment to form a precursor genome, the precursor genome being the
segment or a recombinant of the segment and a prior precursor
genome, such recombinant being formed by at least one recombinase
acting on the recombination elements associated with the segment,
and wherein said step of repeating comprises repeating said step
(b) with each of said predetermined ordering of said plurality of
said segments.
5. The method of claim 4 wherein one of said segments includes an
origin of replication operable in said host organism.
6. The method of claim 5 wherein each of said segments includes a
selectable marker that permits identification of said host
organisms that contain a recombinant of a segment and a prior
precursor genome.
7. The method of 6 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
8. The method of claim 7 wherein said segments are fragments of a
genome from a donor organism.
9. The method of claim 7 wherein said host organism is a
prokaryotic organism and wherein said donor organism is a
prokaryotic organism.
10. The method of claim 9 wherein said host organism and said donor
organism are each bacteria.
11. The method of claim 7 wherein each of said segments is carried
by a separate large-insert vector.
12. The method of claim 11 wherein said step of removing said host
genome includes selecting for said host organisms that loose said
host genome during cell partitioning.
13. The method of claim 7 wherein each of said segments in said
predetermined ordering contains at least a first recombination
element of a kind that is the same as its immediately preceding
segment and at least a second recombinant element of a kind that is
the same as its immediately succeeding segment, wherein such first
and second recombination elements are different kinds.
14. The method of claim 13 wherein at least one of said
recombination elements is a loxP site and wherein at least one of
said recombinases is a Cre recombinase.
15. The method of claim 7 wherein each of said segments is
associated with a pair of recombination elements comprising a first
kind of recombination element and a second kind of recombination
element.
16. The method of claim 15 wherein each of said segments includes a
selectable marker that permits identification of said host
organisms that contain a recombinant of a segment and a prior
precursor genome.
17. The method of 16 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
18. The method of claim 17 wherein said step of transforming
produces a precursor genome containing a pair of recombination
elements of the same kind and wherein said step of transforming
further includes disabling one of said pair of recombination
elements of the same kind.
19. The method of claim 18 wherein said first kind of recombination
element and said second kind of recombination element are each a
loxP site and said recombinase is a Cre recombinase.
20. The method of claim 4 wherein each of said segments is
associated with at least one recombination element selected from a
plurality of different kinds of recombination elements and wherein
each successive segment in said predetermined ordering is
associated with a different kind of recombination element selected
from the plurality.
21. The method of claim 20 wherein each of said segments includes a
selectable marker that permits identification of said host
organisms that contain a recombinant of a segment and a prior
precursor genome.
22. The method of 21 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
23. The method of claim 22 wherein said step of transforming
produces a precursor genome containing a pair of recombination
elements of the same kind and wherein said step of transforming
further includes modifying one of said pair of recombination
elements of the same kind so that it is reactive with a different
kind of recombination element.
24. The method of claim 23 wherein said host organism is a
prokaryotic organism and wherein each of said segments is carried
by a separate large-insert vector.
25. The method of claim 24 wherein at least one of said
recombination elements is a loxP recombination element and wherein
at least one of said recombinases is a Cre recombinase.
26. A method of forming in a host organism a DNA circle comprising
an ordered plurality of polynucleotides, the method comprising the
steps of: (a) providing an ordered plurality of polynucleotides,
each polynucleotide being associated with one or more recombination
elements; (b) transforming or co-transforming the host organism
with one or more polynucleotides to form a precursor DNA circle,
the precursor DNA circle being a recombinant of the one or more
polynucleotides or a recombinant of a prior precursor DNA circle
and the one or more polynucleotides, such recombinant being formed
by recombination of the recombination elements associated with the
one or more polynucleotides; (c) repeating step (b) with
polynucleotides of the ordered plurality of polynucleotides until
the DNA circle is formed.
27. The method of claim 26 wherein said step of transforming or
co-transforming comprises co-transforming said host organism with
said plurality of said polynucleotides so that said DNA circle is
formed.
28. The method of claim 27 wherein each end of each of said
polynucleotides of said plurality has a unique overlapping sequence
region with an end of another said polynucleotide of said plurality
so that an ordering of said polynucleotides is established in said
DNA circle that corresponds to said ordered plurality.
29. The method of 26 wherein said step of transforming or
co-transforming comprises transforming said host organism with a
polynucleotide to form a precursor DNA circle, the precursor DNA
circle being the polynucleotide or a recombinant of the
polynucleotide and a prior precursor DNA circle, such recombinant
being formed by at least one recombinase acting on the
recombination elements associated with the polynucleotide, and
wherein said step of repeating comprises repeating step (b) with
each of said ordered plurality of said polynucleotides until said
DNA circle is formed.
30. The method of claim 29 wherein one of said polynucleotides of
said ordered plurality includes an origin of replication operable
in said host organism.
31. The method of claim 30 wherein each of said polynucleotides
includes a selectable marker that permits identification of said
host organisms that contain a recombinant of a polynucleotide and a
prior precursor DNA circle.
32. The method of 31 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
33. The method of claim 32 wherein said host organism is a
prokaryotic cell and wherein each of said polynucleotides is
carried by a separate large-insert vector.
34. The method of claim 33 wherein said host organism is a
bacteria.
35. The method of claim 33 wherein two or more of said
polynucleotides of said ordered plurality have a length greater
than 300 kilobases.
36. The method of claim 26 wherein each of said segments is
associated with at least one recombination element selected from a
plurality of different kinds of recombination elements and wherein
each successive segment in said predetermined ordering is
associated with a different kind of recombination element selected
from the plurality.
37. The method of claim 36 wherein each of said segments includes a
selectable marker that permits identification of said host
organisms that contain a recombinant of a segment and a prior
precursor DNA circle.
38. The method of 37 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
39. The method of claim 38 wherein said step of transforming
produces a precursor DNA circle containing a pair of recombination
elements of the same kind and wherein said step of transforming
further includes modifying one of said pair of recombination
elements of the same kind so that it is reactive with a different
kind of recombination element.
40. The method of claim 39 wherein said host organism is a
prokaryotic organism and wherein each of said segments is carried
by a separate large-insert vector.
41. The method of claim 40 wherein at least one of said
recombination elements is a loxP recombination element and wherein
at least one of said recombinases is a Cre recombinase.
42. A composition comprising a modular bacterial genome produced by
the steps of: (a) providing a plurality of genomic fragments that
cover a bacterial genome, each genomic fragment being associated
with one or more recombination elements; (b) transforming a host
cell having a host genome with a genomic fragment to form a
precursor genome, the precursor genome being the genomic fragment
or a recombinant of the genomic fragment and a prior precursor
genome, such recombinant being formed by at least one recombinase
acting on the recombination elements associated with the genomic
fragment; (c) repeating step (b) with each of a predetermined
ordering of genomic fragments until the modular genome is formed;
and (d) removing the host genome.
43. The composition of claim 42 wherein one of said genomic
fragments includes an origin of replication operable in said host
cell.
44. The composition of claim 43 wherein each of said genomic
fragments includes a selectable marker that permits identification
of said host cells that contain a recombinant of a genomic fragment
and a prior precursor genome.
45. The composition of claim 44 wherein said step of transforming
further includes selecting said host cells that are identified as
containing said recombinant by said selectable marker.
46. The composition of claim 45 wherein each of said genomic
fragments is carried by a separate large-insert vector.
47. The composition of claim 46 wherein each of said genomic
fragments is associated with at least one recombination element
selected from a plurality of different kinds of recombination
elements and wherein each successive genomic fragment in said
predetermined ordering is associated with a different kind of
recombination element selected from the plurality.
48. The composition of claim 47 wherein each of said genomic
fragments includes a selectable marker that permits identification
of said host organisms that contain a recombinant of a genomic
fragment and a prior precursor genome.
49. The composition of 48 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
50. The composition of 49 wherein said step of transforming
produces a precursor genome containing a pair of recombination
elements of the same kind and wherein said step of transforming
further includes modifying one of said pair of recombination
elements of the same kind so that it is reactive with a different
kind of recombination element.
51. The composition of claim 50 wherein said host organism is a
prokaryotic organism and wherein each of said genomic fragments is
carried by a separate large-insert vector.
52. The composition of claim 51 wherein at least two or more of
said genomic fragments of said predetermined ordering have a length
greater than 300 kilobases.
53. The composition of claim 52 wherein each of said recombination
elements is a loxP site and said recombinase is a Cre
recombinase.
54. A kit for producing a modular prokaryotic genome, the kit
comprising: an ordered plurality of large-insert vectors, each
large-insert vector being associated with one or more recombination
elements and capable of incorporating an insert and each
large-insert vector containing at least a first recombination
element in common with its immediately preceding large-insert
vector in the ordered plurality and at least a second recombinant
element in common with its immediately succeeding large-insert
vector in the ordered plurality, wherein such first and second
recombination elements are different.
55. The kit of claim 54 further including at least one
recombinase.
56. The kit of claim 55 wherein each of said recombination elements
is a loxP site and said recombinase is a Cre recombinase.
57. The kit of claim 56 wherein each of said large-insert vectors
is selected from the group consisting of BACs, cosmids, and
plasmids.
58. A kit for producing a modular prokaryotic genome, the kit
comprising: an ordered plurality of large-insert vectors, each
large-insert vector being associated with one or more recombination
elements and capable of incorporating an insert and each successive
large-insert vector in the ordered plurality having at least one
recombination element selected from a plurality of different kinds
of recombination elements and wherein each successive large-insert
vector in the ordered plurality is associated with a different kind
of recombination element selected from the plurality.
59. The kit of claim 58 further including at least one
recombinase.
60. The kit of claim 59 wherein at least one of said recombination
elements is a loxP site and said recombinase is a Cre
recombinase.
61. The kit of claim 60 wherein each of said large-insert vectors
is selected from the group consisting of BACs, cosmids, and
plasmids.
62. A host organism containing a DNA circle comprising an ordered
plurality of polynucleotides, the DNA circle produced by the steps
of: (a) providing an ordered plurality of polynucleotides, each
polynucleotide being associated with one or more recombination
elements; (b) transforming or co-transforming the host organism
with one or more polynucleotides to form a precursor DNA circle,
the precursor DNA circle being a recombinant of the one or more
polynucleotides or a recombinant of a prior precursor DNA circle
and the one or more polynucleotides, such recombinant being formed
by recombination of the recombination elements associated with the
one or more polynucleotides; (c) repeating step (b) with
polynucleotides of the ordered plurality of polynucleotides until
the DNA circle is formed.
63. The host organism of claim 62 wherein said step of transforming
or co-transforming comprises co-transforming said host organism
with said plurality of said polynucleotides so that said DNA circle
is formed.
64. The host organism of claim 63 wherein each end of each of said
polynucleotides of said plurality has a unique overlapping sequence
region with an end of another said polynucleotide of said plurality
so that an ordering of said polynucleotides is established in said
DNA circle that corresponds to said ordered plurality.
65. The host organism of claim 62 wherein said step of transforming
or co-transforming comprises transforming the host organism with a
polynucleotide to form a precursor DNA circle, the precursor DNA
circle being the polynucleotide or a recombinant of the
polynucleotide and a prior precursor DNA circle, such recombinant
being formed by at least one recombinase acting on the
recombination elements associated with the polynucleotide, and
wherein said step of repeating comprises repeating said step (b)
with each of said ordered plurality of polynucleotides until said
DNA circle is formed.
66. The host organism of claim 65 wherein one of said
polynucleotides of said ordered plurality includes an origin of
replication operable in said host organism.
67. The host organism of claim 66 wherein each of said
polynucleotides includes a selectable marker that permits
identification of said host organisms that contain a recombinant of
a polynucleotide and a prior precursor DNA circle.
68. The host organism of 67 wherein said step of transforming
further includes selecting said host organisms that are identified
as containing said recombinant by said selectable marker.
69. The host organism of claim 68 wherein said host organism is a
prokaryotic cell and wherein each of said polynucleotides is
carried by a separate large-insert vector.
70. The host organism of claim 69 wherein said host organism is a
bacteria.
71. The host organism of claim 70 wherein two or more of said
polynucleotides of said ordered plurality have a length greater
than 300 kilobases.
72. The method of claim 65 wherein each of said segments is
associated with at least one recombination element selected from a
plurality of different kinds of recombination elements and wherein
each successive segment in said predetermined ordering is
associated with a different kind of recombination element selected
from the plurality.
73. The method of claim 72 wherein each of said segments includes a
selectable marker that permits identification of said host
organisms that contain a recombinant of a segment and a prior
precursor DNA circle.
74. The method of 73 wherein said step of transforming further
includes selecting said host organisms that are identified as
containing said recombinant by said selectable marker.
75. The method of claim 74 wherein said step of transforming
produces a precursor DNA circle containing a pair of recombination
elements of the same kind and wherein said step of transforming
further includes modifying one of said pair of recombination
elements of the same kind so that it is reactive with a different
kind of recombination element.
76. The method of claim 75 wherein said host organism is a
prokaryotic organism and wherein each of said segments is carried
by a separate large-insert vector.
77. The method of claim 76 wherein at least one of said
recombination elements is a loxP recombination element and wherein
at least one of said recombinases is a Cre recombinase.
Description
[0001] This application claims priority from U.S. provisional
applications Ser. Nos. 60/725,630 filed 13 Oct. 2005, which is
hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to methods and compositions
for genetically modifying microorganisms, and more particularly, to
methods and compositions for selectively modifying genomic regions
containing predefined sets of genes and/or genetic elements.
BACKGROUND
[0003] Metagenomics and the development of techniques for the
facile production and manipulation of large fragments of DNA have
spurred interest in engineering microorganisms for a host of
important industrial and medical applications, e.g. Lorenz et al,
Nature Reviews Microbiology, 3: 510-516 (2005); Branda et al,
Developmental Cell, 6: 7-28 (2004); Kodumal et al, Proc. Natl.
Acad. Sci., 101: 15573-15578 (2004); Tian et al, Nature, 432:
1050-1054 (2004). Metagenomics is the application of genomics
techniques to the study of communities of microbial organisms
directly in their natural environments, bypassing the need for
isolation and laboratory cultivation of individual species, e.g.
Reisenfeld et al, Annu. Rev. Genet., 38: 525-552 (2004). Such
studies have revealed a wealth of genes encoding novel biochemical
pathways and biocatalysts that potentially could play important
roles in industrial processes, such as the extraction of fuels from
refractory petroleum deposits, the conversion of agricultural raw
materials into bulk and specialty chemicals, the generation of
fuels from renewable resources, the discovery and development of
therapeutically useful products, and the like, e.g. Handelsman,
Microbiol. Mol. Biol. Rev., 68: 669-685 (2004); Van Hamme et al,
Microbiol. Mol. Biol. Rev., 67: 503-549 (2003); Aitken et al,
Nature, 431: 291-294 (2004). In particular, it is expected that
metagenomics will provide an important source of raw materials for
metabolic engineering, that is, the improvement of cellular
activities by manipulation of enzymatic, transport, and regulatory
functions of cells with the use of recombinant DNA technology,
Bailey, Science, 252: 1668-1674 (1991).
[0004] In parallel with the above developments, there has been
great interest in designing host organisms that are genetically
well defined and, in some sense, minimal, not only for engineering
applications, but also for understanding the basic life processes
of free living microbes, e.g. Koonin, Annu. Rev. Genomics Hum.
Genet., 1: 99-116 (2000); Kolisnychenko et al, Genome Research, 12:
640-647 (2002); Kobayashi et al, Proc. Natl. Acad. Sci., 100:
4678-4683 (2003); Glass et al, Proc. Natl. Acad. Sci., 103: 425-430
(2006); Posfai et al, Science, 312: 1044-1046 (2006); and the like.
The primary approach in such studies has been to compare genomes of
related species or strains to identify common genes and then to
systematically delete genes or elements that are not shared, or to
randomly disrupt genes and measure the effect on organism
viability, e.g. Hutchinson et al, Science, 286: 2165 (1999). While
reduced bacterial genomes appear to provide some advantages for
protein production, the basic genome structure of such organisms
remains unchanged from the essentially unordered assembly of genes,
operons, and gene clusters provided by evolution, in which the
genetic elements involved in every life function are scattered
throughout the genome, Wolf et al, Genome Research, 11: 356-372
(2001); Bentley et al, Annu. Rev. Genet., 38: 771-792 (2004);
Ochman et al, Curr. Opin. Microbiol., 6: 109-113 (2003); Ward et
al, Curr. Opin. Microbiol., 8: 564-571 (2005). Such lack of order
increases the difficulties of manipulating multiple genes and/or
regulatory elements that may be involved in the same metabolic
pathway.
[0005] In view of the above, it would be useful to have available a
host organism for metabolic engineering that had a modular genome
structure that permitted placement of functionally related genes
and genetic elements in common regions and that allowed facile
replacement of such regions, thereby taking advantage of recent
advances in large-fragment polynucleotide synthesis and
manipulation.
SUMMARY OF THE INVENTION
[0006] The invention provides methods and compositions for
assembling large nucleic acid constructs, such as modular
replacement genomes in host microorganisms. After such assembly,
the host organism's genome is inactivated or otherwise removed to
permit full control of host cellular functions by the replacement
genome. A modular replacement genome comprises an assembly of
nucleic acid fragments, or segments, derived from one or more
natural organisms or from synthetic polynucleotides or from a
combination of both. Such an assembly, or set, of segments making
up a replacement genome comprises a substantially complete set of
genes and regulatory elements for carrying out minimal life
functions under predefined culture conditions. "Substantially
complete" in reference to the set of such genes and/or regulatory
elements means that a minority of one or more genes and/or
regulatory elements may be provided by episomal elements, such as
plasmids, or the like, separate from the replacement genome. In one
aspect, genes and regulatory elements of a replacement genome are
selected to be substantially compatible with host genome
counterparts. Such compatibility is usually accomplished by
selecting a host genome-replacement genome pair whose sequences are
sufficiently orthologous that (i) a substantial majority of
replacement genes encode gene products or give rise to metabolites
that do not interfere with host organism growth or replication, or
are not otherwise poisonous to the host organism, and (ii) any
subset of replacement genes giving rise to interfering or poisonous
gene products or metabolites can be placed under inducible
control.
[0007] In one aspect, component fragments, or segments, of a
replacement genome are prepared separately in large-insert vectors
wherein each different fragment is associated with one or more
unique recombination elements. A plurality of such
segment-containing vectors that collectively provide a complete
sequence of a replacement genome is assembled stepwise in a
predetermined order by repeated cycles of transformation of a host
organism. In each successive cycle, a precursor replacement genome
is formed that contains an additional segment, until a complete
replacement genome is formed. Alternatively, a plurality of such
segments may be assembled in parallel or in series by one or more
cycles of co-transformation of a suitable host that permits
self-assembly of multiple segments either by providing multiple
segments with non-cross-reacting site-specific recombination
elements or by the presence of a robust DNA repair system for
joining double stranded breaks, e.g. as in D. radiodurans. After a
complete replacement genome is formed, the host genome is then
removed or rendered inoperable by inactivation, ablation or loss
during cell partitioning, and any genes deleterious to the host are
"turned on" by providing conditions for their induction, thereby
forming a free-living synthetic cell with the replacement
genome.
[0008] In another aspect, a replacement genome is modular in that
after assembly and removal of the host genome, segments of the
replacement genome may be selectively replaced with modified
segments or added to by insertion of new segments using the
recombinant elements left over from its initial assembly.
[0009] In another aspect, the invention provides a method of
assembling a replacement genome in a host organism comprising the
following steps: (a) providing a plurality of segments that cover a
replacement genome, each segment being associated with one or more
recombination elements; (b) transforming or co-transforming the
host organism with one or more segments to form a precursor genome,
the precursor genome being a recombinant of the one or more
segments or a recombinant of a prior precursor genome and the one
or more segments, such recombinant being formed by recombination of
the recombination elements associated with the one or more
segments; (c) repeating step (b) with segments of a predetermined
ordering until the replacement genome is formed; and (d) removing
the host genome. In one embodiment, the step of transforming or
co-transforming may comprise co-transforming a host organism with
the plurality of segments so that the replacement genome is formed
in one step. In another embodiment, each end of each of the
segments of a plurality has a unique overlapping sequence region
with an end of another segment of the plurality so that the
predetermined ordering is established upon formation of the
replacement genome.
[0010] In another aspect, the invention provides a method of
assembling a replacement genome in a host organism comprising the
steps: (a) providing a plurality of segments that cover a
replacement genome, each segment being associated with one or more
recombination elements; (b) transforming the host organism with a
segment to form a precursor genome, the precursor genome being the
segment or a recombinant of the segment and a prior precursor
genome, such recombinant being formed by at least one recombinase
acting on the recombination elements associated with the segment;
(c) repeating step (b) with each of a predetermined ordering of
segments until the replacement genome is formed; and (d) removing
the host genome. In one embodiment, each of the segments is
associated with at least one recombination element selected from a
plurality of different kinds of recombination elements and each
successive segment in the predetermined ordering is associated with
a different kind of recombination element selected from the
plurality. In particular, the successive recombination elements may
be mutant loxP sites that do not cross react. In one approach of
such assembling process the step of transforming may produce a
precursor genome containing a pair of recombination elements of the
same kind, in which case, in one embodiment, the step of
transforming may further include modifying one of said pair of
recombination elements of the same kind so that it is reactive with
a different kind of recombination element, such as the
recombination element of the next segment.
[0011] In another aspect, the invention provides nucleic acid
constructs made by serial site-specific recombination, as well as
host organisms in which they are assembled and replicated.
[0012] In still another aspect, the invention provides kits for
assembling nucleic acid constructs from component polynucleotides,
or segments, in a host organism, such kits comprising an ordered
plurality of large-insert vectors, each large-insert vector being
associated with one or more recombination elements and capable of
incorporating an insert and each large-insert vector containing at
least a first recombination element in common with its immediately
preceding large-insert vector in the ordered plurality and at least
a second recombinant element in common with its immediately
succeeding large-insert vector in the ordered plurality, wherein
such first and second recombination elements are different. In
another embodiment, kits of the invention include an ordered
plurality of large-insert vectors, each large-insert vector being
associated with one or more recombination elements and capable of
incorporating an insert and each successive large-insert vector in
the ordered plurality having at least one recombination element
selected from a plurality of different kinds of recombination
elements and wherein each successive large-insert vector in the
ordered plurality is associated with a different kind of
recombination element selected from the plurality. Such kits may
further include reagents for carrying out recombination reactions
of the assembly process, including at least one recombinase,
buffers, co-factors, and the like.
[0013] The present invention advantageously addresses short comings
of present technology by providing modular genomes having modules
in the size range of several thousand basepairs, e.g. 5-10 kb, to
many tens of thousands of basepairs, e.g. 50-100 kb, which are
amenable to facile replacement, deletion, and/or additions. Such
modules may be synthetic polynucleotides and may be designed for 1)
controlling gene content, e.g. all of the genes in a multi-step
metabolic pathway or numerous interacting or branching pathways may
be contained on a single module, 2) excluding of genes that encode
inhibitors or otherwise undesirable competing enzymes that divert a
host cell from desired metabolic/synthetic processes, 3) modifying
codon usage to maximize or minimize protein production, 4)
modifying regulatory elements, including promoters, enhancers,
repressors, activators, terminators, or the like, to modulate gene
expression, 5) incorporating codons from non-natural amino acids,
thereby making possible entirely new protein functions, 6)
balancing enzymatic and transport activities to optimize fluxes of
substrates, intermediates, and products in metabolic pathways, and
like objectives.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-1G illustrates schematically several methods of
assembling in a host organism donor genome segments into a
replacement genome.
[0015] FIG. 2 illustrates alternating, or iterative, positive
selection for successively using two positive selection markers in
a segment assembly process.
[0016] FIG. 3 is a genetic map of a vector incorporating loxP
recombination elements for incorporating donor genome segments into
a growing precursor replacement genome.
[0017] FIG. 4 illustrates a recombination reaction between two
single mutation loxP sites that results in a mutation-free site and
a double mutation site in the recombinant product.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The practice of the present invention may employ, unless
otherwise indicated, conventional techniques and descriptions of
organic chemistry, molecular biology (including recombinant
techniques), cell biology, biochemistry, and immunology, which are
within the skill of the art. Such conventional techniques include,
but are not limited to, vector construction, microbial host
transformation, selection and application of genetic markers,
manipulation of large polynucleotide fragments, preparation of
synthetic polynucleotides, application of recombination systems,
nucleic acid sequencing and analysis, and the like. Specific
illustrations of suitable techniques can be had by reference to the
example herein below. However, other equivalent conventional
procedures can, of course, also be used. Such conventional
techniques and descriptions can be found in standard laboratory
manuals such as Genome Analysis: A Laboratory Manual Series (Vols.
I-IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory
Manual, PCR Primer. A Laboratory Manual, and Molecular Cloning: A
Laboratory Manual (all from Cold Spring Harbor Laboratory Press),
Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,
"Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press,
London, Nelson and Cox (2000), Lehninger, Principles of
Biochemistry 3.sup.rd Ed., W. H. Freeman Pub., New York, N.Y. and
Berg et al. (2002) Biochemistry, 5.sup.th Ed., W. H. Freeman Pub.,
New York, N.Y., Casali et al, editors, E. Coli Plasmid Vectors:
Methods and Applications (Humana Press, Totowa, N.J., 2003), all of
which are herein incorporated in their entirety by reference for
all purposes.
[0019] In one aspect, the invention provides a method of
constructing in a host cell a microbial genome by assembling in a
predetermined order polynucleotide segments. The segments may be
synthetic or may be derived from one or more donor organisms. In
one aspect, such assembling is carried out by use of a
recombination system that permits pairs of polynucleotides to be
combined, or linked to one another, to form a single larger
polynucleotide of a predetermined structure. Usually, members of
such pairs are each double stranded replicatable polynucleotides,
such as plasmids, phages, cosmids, BACs, or the like, or comprise
portions of such replicatable polynucleotides. A recombination
system comprises one or more recombinases and one or more
recombination elements, or nucleic acid sequences that are
specifically recognized by a recombinase and that serve as the
joining sites between a pair of polynucleotides. Usually, reactions
using a recombination system take place within a host cell. In some
cases, a reaction using a recombination system may generate
multiple products from which a desired product is selected, e.g. by
selectable markers, size separation, or the like. In accordance
with the invention, a sequence of recombination reactions is
carried out such that in each successive reaction one member of a
pair of polynucleotides to be combined is a product of the previous
reaction. In certain embodiments, the resulting product is referred
to as a "precursor replacement genome." Thus, by such a sequence of
reactions, polynucleotide segments making up a replacement genome
are assembled segment by segment until a replacement genome is
completed. A recombination system is selected so that each
different segment is associated with one or more unique
recombination elements thereby allowing each segment to be
assembled in a predetermined order without interfering with
previously assembled segments. Preferably, the same recombination
system may be used after assembly to selectively replace, delete
from, add to, or otherwise alter, segments of a completed
replacement genome.
[0020] In one aspect of the invention, a plurality of segments are
sequentially assembled to form a replicating nucleic acid construct
inside of a host cell, wherein the size of the construct is larger
than the expected size of conventionally handled genomic DNA, for
example, that handled by conventional laboratory operations, such
as, pipeting, mixing, stirring, transforming, and the like. In
another aspect, the size of such construct is at least 500
kilobases (kb), or at least 600 kb, or at least 700 kb, or at least
800 kb. Typically, such constructs are not replacement genomes
themselves, but may be precursor replacement genomes. In still
another aspect, a plurality of segments collectively comprises a
complete copy of a replacement genome; that is, the plurality of
segments covers the replacement genome. Such coverage may or may
not be redundant in that sequences of some segments of the
plurality may overlap. In other aspects, such coverage is
non-redundant in that there is no overlap among the sequences of
segments in the plurality. A replacement genome may be derived
directly from a genome of a natural strain or species of
microorganism, which is referred to herein as a "donor genome." A
donor genome may be natural or unmodified, or it may have been
modified to add, delete, substitute, or otherwise alter, genes,
regulatory elements, operons, or other elements. Usually, segments
in a plurality that comprise a donor genome are selected so that
they collectively contain a substantially complete sequence of the
donor genome and so that overlap is minimized; that is, each
segment does not include any sequences that overlap with sequences
of any other segment of the plurality, or if such overlap occurs,
then it is minimal, for example, less than ten percent of either
segment, or less than five percent of either segment, or less than
two percent of either segment, or less than one percent of either
segment. In other words, such segments, in some sense, represent a
minimal tiling path of the donor genome. "Substantially complete"
in reference to a set of segments derived from a donor genome means
that the set contains sequences of the donor genome necessary for
growth and replication under defined culture conditions. Thus,
"substantially complete" includes situations where less than the
entire genome sequence is included. In one aspect, "substantially
complete" means at least 85 percent of a donor genome, or at least
90 percent of a donor genome, or at least 95 percent of a donor
genome, or at least 98 percent of a donor genome. When a plurality
of segments making up a donor genome is assembled in accordance
with the invention it is referred to herein as a replacement
genome. A replacement genome derived from a donor genome may be
tailored later by selectively replacing segments containing the
natural sequence components with synthetic polynucleotides in order
to rearrange the ordering and/or composition of genes, regulatory
elements, operons, gene clusters, and the like, for the purpose of
enhancing performance of, or adding new functionality to, the
resulting synthetic organism.
[0021] In some cases, during initial cycles of an assembly process,
replication of a precursor replacement genome in a host organism
may occur by operation of an origin of replication from a
large-insert vector used in its construction, whereas replication
in later cycles is controlled by an origin of replication derived
from a donor genome. In other cases, immediately after or within
the first few cycles of assembly, e.g. 1-2, or 2-4, or 3-6 assembly
cycles, replication of a precursor replacement genome occurs by
operation of an origin of replication derived from a donor genome.
In such embodiments, early assembled segments, e.g. segments 1 to
4, or so, contain sequences comprising the donor origin of
replication. In either case, a host is preferably selected so that
the nucleic acids and proteins responsible for replication are
substantially cross-functional with those encoded by the donor
genome. That is, host organism proteins responsible for replication
are operable with the donor origin of replication. In some
embodiments, donor transcription factors may be added, e.g. via a
host plasmid, to ensure that necessary proteins for precursor
replacement genome replication are available. In some cases, other
donor transcription factors may be provided to ensure availability
of other necessary proteins, e.g. a particular donor replication
protein for which there is no cross-functional host protein, or the
like. Accordingly, in one aspect, preferably, a segment early in
the assembly process (e.g. cycle 1-2, or 1-4, or 1-6) contains the
replacement genome origin of replication (which may be a donor
genome origin of replication) and all genes encoding necessary
replication factors or proteins.
[0022] Likewise, whenever a donor genome contains genes that encode
products that are incompatible with host organism growth and
replication, then such genes, if indispensable, may be moved to the
final segment in the assembly process; otherwise, if dispensable,
such genes may be deleted. Optionally, such genes may be placed
under inducible control, so that they may be activated after
assembly is complete and the host genome and associated support
functions are no longer required. In one aspect, donor genes (or
other sequences) incompatible with a host organism can be
identified by whole genome shotgun sequencing using conventional
techniques, e.g. Weber et al, Genome Research, 7: 401-409 (1997);
Adams et al, Science, 287: 2185-2195 (2000); Waterston et al, Proc.
Natl. Acad. Sci., 99: 3712-3716 (2002); Reed et al, J. Virol.
Meth., 129: 91-96 (2005); and the like, which references are
incorporated by reference. Briefly, in whole genome shotgun
sequencing, genomic DNA of a donor organism is randomly sheared,
cloned in small-, medium-, and large-insert expression vectors,
transformed into the host organism, and randomly selected clones
are sequenced. A sufficient amount of sequencing is carried out so
that if gaps remain in the assembled donor genome, it must be
attributed to the presence of a cloned sequence that is
incompatible with the host organism, and not with under sampling of
donor genome fragments. The incompatible donor sequences are
identified by the locations of the gaps in the assembled donor
genome. Alternatively, or as a confirmation, individually selected
donor sequences may be tested by introducing into a host in a
suitable expression vector.
[0023] In one aspect, a host organism and donor organism are
selected so that proteins responsible for replication are
substantially cross-functional, thereby allowing replication of
precursor replacement genomes by host replication proteins. One
approach to accomplish this is selection of host organisms and
donor organisms that are close evolutionarily, e.g. as measured by
genome sequence homology, particularly of core function genes, e.g.
those involved with replication, transcription, protein synthesis,
substrate transport, energetic metabolism, cell division, and the
like. Guidance for selecting core function genes for determining a
measure of cross-functionality of encoded proteins may be found in
an extensive literature on minimal microbial genomes, e.g. as
represented by the following references that are incorporated by
reference: Koonin, Annu. Rev. Genomics Hum. Genet., 1: 99-116
(2000); Kobayashi et al, Proc. Natl. Acad. Sci., 100: 4678-4683
(2003); Glass et al, Proc. Natl. Acad. Sci., 103: 425-430 (2006);
Gil et al, Microbiol. Molecular Biol. Rev., 68: 518-537 (2004);
U.S. Pat. No. 6,673,567; U.S. Pat. No. 6,207,384; and the like. In
one aspect, core function genes of host and donor genomes are at
least 50 percent homologous; in another aspect, core function genes
of host and donor genomes are at least 60 percent homologous; or at
least 70 percent homologous, or at least 80 percent homologous, or
at least 90 percent homologous, or at least 95 percent homologous,
or at least 98 percent homologous. In one aspect, core function
genes for determining cross-functionality are genes encoding
proteins necessary for genome replication. Whenever Escherichia
coli (E. coli) is employed as a host organism, core function genes
for genome replication are well known to those of ordinary skill in
the art, as evidence by the treatise: Komberg and Baker, DNA
Replication, Second Edition (Freeman, San Francisco, 1992), which
is incorporated by reference. In one aspect, such genes include
those encoding polymerases, primases, ligases, helicases, and
gyrases. In another aspect, such genes are selected from the
following set: dnaA, dnaB, dnaC, dnaE, dnaG, dnaJ, dnaK, dnaN,
dnaQ, dnaT, dnaX, dnaY, dnaZ, dut, grpE, gyrA, gyrB, lig, nrdA,
nrdB, ori, polA, polB, priA, priB, priC, rep, mhA, rpoA, rpoB,
rpoC, rpoD, ssb, ter, topA, trxA, and tus, where these gene
designations are defined in Kornberg and Baker (cited above), and
are defined by sequence in publicly available databases, such as
NCBl, Ensembl, GenBank, or the like. In particular, the sequence of
E. coli strain K-12 is disclosed in Blattner et al, Science, 277:
1453-1474 (1977), which is incorporated by reference. Whenever
Bacillus subtilis is employed as a host organism core function
genes for genome replication may be selected from the following
set: dnaA, dnaB, dnaC, dnaD, dnaE, dnaF, dnaG, dnaH, dnaI, dnax,
gyrA, gyrB, nrdA, and the like.
[0024] Alternatively, activities of selected host proteins in
carrying out donor functions may be assessed empirically where
assays are available, e.g. a donor origin of replication may be
cloned into a host using a vector whose own origin of replication
is under conditional control, e.g. temperature sensitive control,
so that upon disablement of the vector origin, cross-functionality
may be tested by assessing whether and to what extent the vector
replicates (by use of host replication proteins).
[0025] Replacement genomes may be assembled in accordance with the
invention in a wide variety of host organisms. Preferably, the host
organism is a prokaryotic organism. In one aspect, the host
organism is a bacterium, and more usually, an enteric bacterium,
such an E. coli. In another aspect, the host organism is a Bacillus
subtilis. Preferably, the donor organism is a prokaryotic organism.
In one aspect, the donor organism is a bacterium. Exemplary donor
organisms for use with an E. coli host include Hemophilus, and more
particularly, Hemophilus influenzae, Pseudomonas, and more
particularly, Pseudomonas putida.
[0026] In one aspect, segments used in assembling a replacement
genome are cloned or constructed using conventional techniques in
conventional cloning vectors, including plasmids, phages, cosmids,
and/or bacterial artificial chromosomes (BACs) and P1-derived
artificial chromosomes (PACs), P1 vectors, and the like. In order
to minimize assembly steps, preferably, most, if not all, segments
are provided as inserts of large-insert cloning vectors, such as
BACs or PACs. After assembly of a replacement genome is completed,
the type of vector used and the sizes of replacement fragments for
further alterations will depend on particular applications. A
large-insert vector is a vector capable containing an insert having
a length in the range of from 50 kb to 300 kb, or greater, and
transforming a prokaryotic host organism, such as a bacteria. In
particular, a large number of BACs are available for use in
RecA.sup.- E. coli host organisms. In one aspect, a set of segments
for assembling a replacement genome may comprise inserts in the
same type of cloning vector or in different types of cloning
vectors, e.g. a majority may comprise large fragments, i.e. greater
than 100 kb, in BACs and a minority may comprise smaller fragments
in other cloning vectors, such as phages, comids and/or plasmids.
In another aspect, BACs are employed as the primary cloning vector
for segments of a replacement genome; that is, a majority of the
sequence of a replacement genome is provided in BACs. Lengths of
segments in a plurality may vary widely depending on several
factors including the size of a donor genome, the desirability of
minimizing steps in the assembly process, the desired arrangement
of genes, operons, gene clusters and the like, anticipated segment
substitutions (for example, it may be more efficient to use several
smaller synthetic segments separately rather than a single large
synthetic segment), and so on. In one aspect, segments have lengths
in the range of from a few thousands of basepairs, e.g. 2-10 kb, to
several hundreds of thousands of basepairs, e.g. 100-300 kb. In
another aspect, segments have lengths in the range of from tens of
thousands of basepairs, e.g. 10-50 kb, to hundreds of thousand
basepairs, e.g. 100-300 kb. Preferably, segments are cloned in BAC
vectors, which are described in the following references that are
incorporated by reference: Zhao et al, editors, Bacterial
Artificial Chromosomes (Humana Press, Totowa, N.J., 2004); Kim et
al, Genomics, 34: 213-218 (1996); Shizuya et al, Proc. Natl. Acad.
Sci., 89: 8794-8797 (1992); U.S. Pat. Nos. 5,874,259 and 6,472,177;
and the like. Techniques for assembling inserts into BACs from
several smaller pieces are well known in the art, as evidenced by
the following reference: O'Connor et al, Science, 1307-1312 (1989),
which is incorporated by reference. Exemplary vectors that may be
used with the invention, with no or minor modifications, include
pBeloBAC11, pBACe3.6, pCC1BAC, pSMART VC, pIndigoBAC-5, SuperCos 1,
and the like, which are commercially available or described in
GenBank.
[0027] Assembly of a replacement genome may be carried out using a
variety of techniques, including the use of restriction
endonucleases and ligases for inserting fragments, the use of hosts
having robust DNA repair mechanisms, and the use of recombination
based methods for site-specific insertion. Site-specific
recombination systems for use with the invention include at least
one recombinase that usually operates on a pair of reactive
recombination elements (or sites) to catalyze strand scission and
rejoining. Reactive recombination elements are usually, but not
necessarily, recombination sites having identical sequences.
Recombination systems may also include additional ancillary
proteins that may be operationally associated with a recombinase.
In one aspect, in order to sequentially assemble different segments
into a growing precursor replacement genome, a recombinase is
selected that is capable of catalyzing separate recombination
events with recombination elements having different sequences
without the occurrence of significant cross reaction among
different recombination elements. Thus, in one approach, a
sufficient number of different non-cross reacting recombination
elements must be available for complete assembly of a replacement
genome. In another approach, non-cross reacting recombination
elements may be re-used in alternating steps of assembly; thus,
only two non-cross reacting recombination elements are required.
Many recombination systems are useful in the present invention and
may be used alone or in combination with one another. Suitable
recombination systems include, but are not limited to: 1) linear
homologous recombination using two crossover sites near the ends of
the sequence of interest, exemplified by a Red/ET system; 2) circle
homologous integration followed by a second resolving
recombination, exemplified by Cre-10.times. or flp-frt sites in a
recombination mediated cassette exchange (RMCE) approach; 3)
linear, sequence-specific recombination (e.g., via a phage
integrase such as .lamda. or phiC31); and 4) sequence-specific
circle integration. Exemplary site-specific and homologous
recombination systems include, but are not limited to, Cre-loxP,
Flp-FRT, att-Int (Gateway), Red/ET, RecA, and the like. These and
other recombination systems are well-known to those of ordinary
skill in the art and are described in the following references,
which are incorporated by reference: Branda et al, Developmental
Cell, 6: 7-28 (2004); Baer et al, Curr. Opin. Biotech., 12: 473-480
(2001); Sauer, Nucleic Acids Research, 24: 4608-4613 (1996); Yu et
al, Proc. Natl. Acad. Sci., 97: 5978-5983 (2000); Lee et al,
Genomics, 73: 56-65 (2001); Muyrers et al, EMBO Rep., 1: 239-243
(2000); Cheo et al, Genome Research, 14: 2111-2120 (2004);
Missirlis et al, BMC Genomics, 7: 73 (2006); U.S. Pat. Nos.
6,509,156; 6,465,254; 6,720,140; 5,776,449; 5,888,732; and the
like. Recombinases may be provided by expression of genes that may
be carried by the host genome, or by an episome, such as a plasmid,
or by one or more segments of a precursor replacement genome.
Preferably, expressions of recombinases are under inducible control
in order to minimize the occurrence of spurious or undesired
recombination during the assembly process. Also, preferably, a host
organism is selected that is free of recombination elements used in
the replacement genome (or DNA circle) assembly process, or a
selected organism is treated to remove or disable such elements to
prevent spurious or unintended recombination reactions.
[0028] In one aspect, the assembly process of the invention
includes successive steps of recombining in a host organism a new
segment of a replacement genome with segments that have previously
been assembled, and which constitute a precursor replacement
genome. Such steps are carried out using conventional vectors and
transformation techniques in conjunction with a recombination
system, such as one of those indicated above. Typically, each such
step includes substeps of transforming the host with a vector
containing a new segment operationally associated with one or more
unique recombination elements, culturing transformed host
organisms, and selecting host organisms containing recombinants,
i.e., precursor replacement genomes that have successfully
recombined with a new segment to generate a successive precursor
replacement genome (or a completed replacement genome), as the case
may be. In some embodiments, multiple segments may be recombined
with a precursor replacement genome in a single cycle, e.g. using
the approach of Church et al, International patent publication WO
2006/055836, which is incorporated herein by reference.
[0029] In one aspect of the invention, segment assembly is carried
out with site-specific recombination, as illustrated in FIGS.
1A-1G. Site-specific recombination elements are selected and
arranged in vectors to drive recombination reaction to the desired
products. Sets of site-specific recombination elements are provided
that (i) have substantially no cross reactivity with one another,
and (ii) are oriented so that stable integration occurs in each
step of the assembly process. As reviewed by Branda et al (cited
above), for example, the .lamda. integrase family of site-specific
recombination elements, which include loxP and FRT, share a common
mechanism of DNA recombination that involves strand cleavage,
strand exchange, and ligation. Although distinct at the nucleotide
level, loxP and FRT sites share an overall structure which includes
two 13 basepair palindromic sequences, or inverted repeats,
separated by an 8 basepair asymmetric core, or spacer, sequence. In
the presence of two sites, recombinase monomers bound to the
inverted repeats promote the formation of a synaptic complex and
recombination between the two sites. Strand cleavage, exchange, and
ligation occur within the spacers. Because of spacer asymmetry,
strand exchange is possible only when target sites are connected by
synapses in one orientation. Consequently, the relative orientation
of target sites with respect to one another determines the outcome
of recombination: Cre and Flp recombinases will excise a circular
molecule from between two directly repeated target sites, integrate
a circular molecule into a linear molecule each possessing a target
site, invert the DNA between two inverted sites, and exchange
sequences distal to target sites present on two linear molecules.
Because insertion ordinarily leaves two identical sites in cis
configuration, which are themselves substrates for recombination,
stable insertions are difficult or impossible using two wild type
sites. However, many recombinases, such as Cre and Flp, tolerate
certain variations in their target sequences and effectively
catalyze recombination only between certain subsets of the
alternative sites. This property is exploited to permit successive
recombination events for replacement genome assembly. Variant
target sites for .lamda. intergrase recombinases, such as Cre and
Flp, fall into two classes: spacer variant and invert-repeat
variants. The first class contains nucleotide substitutions within
the spacer sequence and exploits the finding that it is spacer
length, not sequence that is the critical factor for efficient
recombination, so long as the sequence between participating sites
is identical. Recombination is therefore efficiently mediated
between pairs of homotypic (e.g. FRT/FRT or F.sub.3/F.sub.3) but
not heterotypic (e.g. FRT/F.sub.3) sites. The second class of
alternative sites (inverted repeat variants) may also be exploited
to provide stable insertions. A target site containing a nucleotide
substitution in the "left side" inverted repeat (an "LE" mutant
site) can recombine with a site containing an analogous
substitution in the "right end" inverted repeat (an "RE" mutant
site), although at a slower reaction rate than wild type sequences.
Such mutants are designed so that the recombination product harbors
one wild type site and one LE/RE double mutant site, the latter
being effectively inert. Thus, insertion with such single mutant LE
and RE sites results in the formation of only one potentially
active recombination element, which itself may be inactivated or
modified and used for subsequent insertions. These concepts are
illustrated in FIGS. 1A-1G for several embodiments of the
invention.
[0030] FIG. 1A illustrates a plurality of segments (100) carried in
vectors, lox1 through loxK, that each have a unique combination of
site-specific recombination elements labeled "A.sub.01,"
"A.sub.10," "B.sub.01," "B.sub.10," "C.sub.01," . . . "K.sub.10,"
where each different letter, "A," "B," etc., indicates a different
non-cross-reacting site-specific recombination element, and where
subscripts "01" and "10" indicate a recombination element has an RE
mutant site ("01") or an LE mutant site ("10"). Correspondingly, a
letter with subscripts "11" indicates a double mutant site and a
letter with subscripts "00" indicates a mutant-free site. It is
noted that the diagrams of vectors are only symbolic
representations and are not to scale or proportion. For example,
even though the site-specific recombination elements are shown at
opposite sides of the vectors, this is not a required
configuration. The recombination elements may be juxtaposed or they
may be interspersed in the vector or segment. Vector lox1 (101)
containing initial segment (102) requires only a single
recombination element "A.sub.01" (104) in this embodiment.
Likewise, vector loxK (106) containing the final segment (108)
requires only a single recombination element "K.sub.10" (110).
Vectors lox2 through loxK-1 each have two different recombination
elements, as exemplified by vector lox4 (112), which comprises
segment (114), recombination element "C.sub.10" (116),
recombination element "D.sub.01" (118), and portion (120), which
may be part of segment (114) or simply a connection between the two
recombination elements (e.g. a bond connecting two adjacent
sequences or an intervening polynucleotide). In this embodiment,
each of the segments-containing vectors of plurality (100), except
for the first (lox1) and the last (loxK), contains at least a first
recombination element (e.g. "A.sub.10" of lox2) in common with its
immediately preceding vector (i.e. "A.sub.01" of lox1) in the
predetermined order shown and at least a second recombinant element
(e.g. "B.sub.10" of lox2) in common with its immediately succeeding
vector (i.e. "B.sub.10" of lox3), wherein such first and second
recombination elements are different (i.e. in this embodiment, the
first is type "A" and the second is type "B"). Again, an important
property of the different types (or kinds) of recombination
elements is that members or variants of one type (or kind) do not
cross react (or substantially do not cross react) with members or
variants of another type (or kind). As illustrated in FIG. 1B,
segments of plurality (100) are assembled stepwise by adding them
on segment at a time to form a succession of precursor replacement
genomes. In one aspect, each step in the assembly process comprises
a cycle of steps (or substeps) including transforming a host and
selecting a resulting transformant using a selectable marker. The
relative ordering of recombination elements is shown in first
recombinant (122), which is the first precursor replacement genome.
Since site-specific recombination is conservative, in that DNA
synthesis is not required and sequences are neither lost nor gained
in the reaction, first recombinant (122) contains pieces of all the
recombination elements of the two vectors that were combined, i.e.
two copies of an "A" type recombination element in double mutant
form (104) and in mutant-free form (105), and one copy of
recombination element "B.sub.01" (124), which serves as the unique
recombination site for the next vector, lox3. After transformation,
recombination and selection, the resulting host harboring first
recombinant (122) is transformed with vector lox3 to form the next
recombinant, or precursor replacement genome (126). Again,
sequences are conserved in the recombinant of (122) and lox3 so
that precursor replacement genome (126) contains five recombination
elements: "A.sub.00" (active), "A.sub.11" (inert), "B.sub.00"
(active), "B.sub.11" (inert), and "C.sub.01" (128), which again is
the unique recombination site for the next vector, lox4. The
process continues until a replacement genome is complete. The
ordering of the active recombination elements ("A.sub.00,"
"B.sub.00," "C.sub.00," etc.) relative to the inert recombination
elements ("A.sub.11," "B.sub.11," "C.sub.11," etc.) may be varied
by changing the ordering of the LE and RE mutant sites in vectors
(100). For example, if lox2 contained "B.sub.10" and lox3 contained
"B.sub.01," then the positions of "B.sub.11" and "B.sub.00" would
be swapped and the resulting vector corresponding to (126) would
have inert site "A.sub.11" sandwiched between "A.sub.00" and
"B.sub.00.". As indicated, in this embodiment, recombination takes
place in a host cell, such as illustrated diagrammatically in FIG.
1C. Host organism (130) is transformed by initial vector (101) to
form a host containing a host genome (132) and vector (101). In
subsequent cycles of the assembly process, successively larger
recombinants (134), i.e. precursor replacement genomes, are formed
until a completed replacement genome (136) is present. Host genome
(132) is then removed or ablated to give synthetic cell (138)
containing only replacement genome (136).
[0031] As discussed more fully below, the above process may be
carried out with pairs of LE and RE mutant recombination elements
for each type, "A" through "K," as taught by Missirlis et al, BMC
Genomics, 7: 73 (4 Apr. 2006), which is incorporated by reference.
Briefly, LE and RE mutant pairs are prepared for each type of
recombination element. When a recombination event occurs (e.g.,
part of element "B10" on lox3 is combined with element "B01" on
lox3), both mutants are present in only one of the product sites,
and the other product site is free of mutations. This results
directly in a modular replacement genome. That is, the operable
recombination sites may be used with the recombination system
employed to exchange segments for modifying the properties of the
synthetic organism, e.g. using a RMCE procedure.
[0032] In another aspect, segments may be assembled into a
replacement genome by using fewer recombinations elements, as
illustrated in FIGS. 1D and 1E. A plurality of segments is provided
in vectors V.sub.1 through V.sub.K (150). In this embodiment, the
vectors do not each have one or more unique recombination elements;
instead, the recombination elements are re-used in alternating
cycles of segment incorporation. Such re-cycling of recombinIation
elements may be accomplished with a plurality of different types of
recombination elements, each of which is provided as a pair of
single mutants that may recombine with each other to produce an
active mutant-free form and an inert double mutant form. Such a
plurality of different recombination elements may contain two,
three, four, five, six, seven, eight recombination elements. In one
aspect, recombination elements may be conveniently introduced into
BACs carrying the segments by Red/ET recombination, e.g. as
disclosed in U.S. Pat. No. 6,509,156; and Yu et al, Proc. Natl.
Acad. Sci., 97: 5978-5983 (2000); and/or using reagents
commercially available from GeneBridges GmbH (Dresden, Germany). In
particular, using appropriate host bacteria, sequences to be
inserted into a BAC may be prepared by PCR, where the resulting
amplicon contains unique flanking sequences of 30-50 basepairs.
Such amplicons are recombined with regions of the BAC bounded by
the same unique sequences.
[0033] In FIG. 1D, vector V.sub.1 containing the first segment and
vector V.sub.K containing the last segment, V.sub.K, each have a
single recombination element, and the rest of the vectors, V.sub.2
through V.sub.K, .sub.1, each have two. Recombination element
A.sub.01 (152) on V.sub.1 is a single mutant site that is operable
with recombination element A.sub.10 (154) on V.sub.2, which is a
different single mutant site. Likewise, recombination element
B.sub.01 (156) on V.sub.2 is a single mutant site that is operable
with recombination element B.sub.10 (158) on V.sub.3, which is a
different single mutant site. The same four sites may be used with
all of the vectors V.sub.1 through V.sub.K, when used as follows.
V.sub.1 and V.sub.2 are transformed into a Red/ET competent host
that also expresses an appropriate recombinase to form recombinant
(160), in which recombination elements A.sub.01 (V.sub.1) and
A.sub.10(V.sub.2) are changed to functional A.sub.00 site (162) and
non-functional A.sub.11 site (164). An amplicon is prepared
containing 30-50 basepair flanking sequences that are identical to
sequences flanking A.sub.00 (162) on recombinant (160). The host
bacteria containing recombinant (160) is transformed with the
amplicon so that it can recombine (166) with the portion of
recombinant (160) containing A.sub.00 (162) to produce recombinant
(169), which is shown to have a disabled recombination site "X"
(168). An advantage of the Red/ET system is that recombinants can
be detected by PCR; growth on a selective medium is not required.
Modified recombinant (169) may then be used in the next assembly
step by transforming its host with vector V.sub.3 containing a
third segment and recombination elements B.sub.10 and A.sub.01 to
form (171) recombinant (179), which contains a functional B.sub.00
site and a non-functional B.sub.11 site. As above, after selection
of a recombinant (179), the functional B.sub.00 site is disrupted
(176) to form modified recombinant (or precursor replacement
genome) (180). Assembly of a replacement genome continues in a
similar manner for the remaining segments.
[0034] A recombination system, such as Red/ET may also be used as
illustrated in FIGS. 1F-1G to modify an undesired functional
recombination element within a segment-addition cycle. A plurality
of vectors (180) is provided that each contains only one single
mutant form of one type of recombination element. As above, only
two types recombination elements are shown in the embodiment of
FIG. 1F (A's and B's); however, further types of recombination
elements may be employed in alternative embodiments. Vectors
V.sub.1 and V.sub.2 are recombined to form recombinant (182) that
contains active recombination element Aoo (184) and inert
recombination element A.sub.11 (186). In this embodiment, instead
of inactivating element A.sub.00, a homologous recombination
system, such as Red/ET, is employed to exchange the active
recombination element A.sub.00 with a recombination element
complementary to the element of the next vector to be inserted. (As
above, this allows the type A recombination elements to be re-used
in subsequent steps). After such exchange (188), precursor
replacement genome (190) is formed that has one inactive
recombination element A, (192) and one active recombination element
B.sub.01 (194). Precursor replacement genome (190) is then combined
with vector V.sub.3 so that element B.sub.01 recombines (195) with
element B.sub.10 of vector V.sub.3 to form precursor replacement
genome (196) containing active B.sub.00 (198) and inert B.sub.11
(199). In the next step, B.sub.00 is exchanged (1901) with A.sub.01
to produce precursor replacement genome (1902). Similar cycles
(1904) of transforming to add a segment and transforming to
exchange a recombination element are carried out until a
replacement genome is assembled.
[0035] As mentioned above, assembly of nucleic acid constructs, DNA
circles, or replacement genomes may also be carried out in part or
wholly in parallel by partial or complete self-assembly after
co-transformation. In one aspect of this embodiment, multiple
segments are co-transformed into a host organism wherein each
segment is associated with a site-specific recombination element
that does not cross-react with its co-transforming segments, so
that recombination results in correctly ordered segments in the
nucleic acid construct produced, whether it is a precursor
replacement genome or a completed replacement genome. Segments may
contain multiple independent selective markers so that successful
co-transformants can be identified. Suitable non-cross reacting
recombination elements may be selected from the group of mutant
loxP recombination elements disclosed below. In another aspect of
this embodiment, a plurality of segments having unique overlapping
ends of identical or homologous sequences (i.e. each overlapping
region comprises unique sequences, or at least distinct from other
overlapping regions) may be assembled in parallel by
co-transforming into a suitable host having a robust DNA repair
system, such as Deinococcus radiodurans, which has been studied for
potential applications in bioremediation of radioactively
contaminated environments, e.g. Zahradka et al, Nature, 443:
569-573 (2006); Makarova et al, Microbiol. Mol. Biol. Rev., 65:
44-79 (2001); Brim et al, Nature Biotechnology, 18: 85-90 (2000);
Langer et al, Nature Biotechnology, 16: 929-933 (1998); Narumi et
al, U.S. Pat. No. 6,770,476; which references are incorporated
herein by reference. Suitable overlapping sequences may have
lengths of from a few hundred basepairs, e.g. 100-1000, to several
thousand basepairs, e.g. 1000-20,000. In still another embodiments,
a plurality of segments having unique overlapping ends of identical
or homologous sequences as above may be assembled in parallel by
co-transforming into a suitable host having a homologous
recombination system that recombines the overlapping ends, such as
a Red/ET system, or the like.
[0036] Generally, and in the particular examples above,
transforming host microorganisms with vectors carrying segments is
carried out with conventional techniques. As used herein, the terms
"transformation" and "transfection" are intended to refer to a
variety of art-recognized techniques for introducing an exogenous
nucleic acid sequence (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride co-precipitation,
DEAB-dextran-mediated transfection, lipofection, electroporation,
optoporation, mechanical injection, biolistic injection, and the
like. Suitable methods for transforming or transfecting host cells
are found in Sambrook, et al. (Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989), and like
laboratory manuals.
[0037] Transformed microorganisms, that is, those containing
recombinant molecules, may be selected with a variety of positive
and/or negative selection methods or markers. In certain aspects,
the positive selection marker is a gene that allows growth in the
absence of an essential nutrient, such as an amino acid. For
example, in the absence of thymine and thymidine, cells expressing
the thyA gene survive, while cells not expressing this gene do not.
A variety of suitable positive/negative selection pairs are
available in the art. For example, various amino acid analogs known
in the art could be used as a negative selection, while growth on
minimal media (relative to the amino acid analog) could be used as
a positive selection. Visually detectable markers are also suitable
for use in the present invention, and may be positively and
negatively selected and/or screened using technologies such as
fluorescence activated cell sorting (FACS) or microfluidics.
Examples of detectable markers include various enzymes, prosthetic
groups, fluorescent markers, luminescent markers, bioluminescent
markers, and the like. Examples of suitable fluorescent proteins
include, but are not limited to, yellow fluorescent protein (YFP),
green fluorescence protein (GFP), cyan fluorescence protein (CFP),
umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin
and the like. Examples of suitable bioluminescent markers include,
but are not limited to, luciferase (e.g., bacterial, firefly, click
beetle and the like), luciferin, aequorin and the like. Examples of
suitable enzyme systems having visually detectable signals include,
but are not limited to, galactosidases, glucorinidases,
phosphatases, peroxidases, cholinesterases and the like. In other
aspects, the positive selection marker is a gene that confers
resistance to a compound which would be lethal to the cell in the
absence of the gene. For example, a cell expressing an antibiotic
resistance gene would survive in the presence of an antibiotic,
while a cell lacking the gene would not. For instance, the presence
of a tetracycline resistance gene could be positively selected for
in the presence of tetracycline, and negatively selected against in
the presence of fusaric acid. Suitable antibiotic resistance genes
include, but are not limited to, genes such as
ampicillin-resistance gene, neomycin-resistance gene,
blasticidin-resistance gene, hygromycin-resistance gene,
puromycin-resistance gene, chloramphenicol-resistance gene and the
like. In certain aspects, the negative selection marker is a gene
that is lethal to the target cell in the presence of a particular
substrate. For example, the thyA gene is lethal in the presence of
trimethoprim. Accordingly, cells that grow in the presence
trimethoprim do not express the thyA gene. Negative selection
markers include, but are not limited to, genes such as thyA, sacB,
gnd, gapC, zwJ, talA, taiB, ppc, gdhA, pgi, Jbp, pykA, cit, acs,
edd, icdA, groEL, secA and the like.
[0038] Selection methods and/or markers may be used efficiently in
a multi-step assembly process, such as called for by the invention,
by employing a pair of selection methods or markers that are
switched, or used reciprocally, between successive recombination
steps, e.g. as taught by O'Connor et al, Science, 244: 1307-1312
(1989); Kodumal et al, Proc. Natl. Acad. Sci., 101: 15573-15578
(2004); or the like. For example, as illustrated diagrammatically
in FIG. 2 a first segment-containing vector (200) is transformed
(211) into a host containing a recombination element (201) embedded
in a gene for a positive selective marker ("Marker 1") (202), e.g.
SacBll. The SacBll gene codes for an enzyme that converts sucrose
to levansucrase, which is toxic to bacterial cells (see Pierce et
al., Proc. Natl. Acad. Sci 89; 2056-2060. 1992). Element (201)
divides marker (202) into two parts (203) and (204). A successive
segment-containing vector (206) is transformed (211) into the host
containing a recombination element (201) complimentary to that in
the prior recombinant, and a second non-complementary recombination
element (205) embedded in a second selectable marker gene ("Marker
2") (208) (for example, the tetracycline resistance gene, which
confers sensitivity to fusaric acid, e.g. Bochner et al., J.
Bacteriology 143; 926-933. 1980). As above, recombination element
(205) divides Marker 2 gene (208) into two parts (209) and (210).
Recombination between clone 1 (200) and vector (206) containing
segment (212) forms clone 2 (219) that has a disrupted marker 1
gene (see the separation of parts (203) and (204)), but a fully
functional tetracycline resistance gene (208) containing
recombination element (205); thus, when plated on solid media
containing sucrose, only recombinant clones will grow. Clone 2
(219) may then be recombined (222) with vector (227) containing (i)
complementary recombination element (205), (ii) marker 1 gene (214)
containing embedded recombination element (215) (which divides
marker 1 into parts (216) and (217)), and segment (218). As above,
recombination element (205) of vector (227) reacts with element
(205) of clone 2 (219) to form clone 3 (231), in which marker 2
(208) of clone 2 is disrupted by the separation of parts (209) and
(210), but which also contain a functional marker (214) that
permits selection of recombinants by exposure to tetracycline.
Functional marker (214) also contains embedded recombination
element (215). Segments (212) and (218) are contained clone 3.
Assembly continues by providing vector (233) that contains the next
segment (224) and a marker 2 gene (225) containing another embedded
recombination element (229) as well as a complementary
recombination element (215) for insertion into clone 3. Vector
(233) and clone 3 (231) form recombinant clone 4 (240) and three
segments, the final one of which (224) disrupts marker 1 gene (214)
(by splitting its coding region, see (216) and (217)) and provides
new operational marker 2 gene (225). Alternating disruptable
positive selection markers in this manner allows stepwise accrual
of donor genome segments in the host organism. A disruptable
positive selection marker may also comprise a recombination element
that is positioned between a marker gene and its promoter site.
[0039] After a replacement genome is assembled and becomes operable
in a host organism, the host genome is removed or ablated. In one
aspect, it is removed by creating conditions that select against
host organisms that retain the host genome. In another aspect,
ablating the host genome includes creating conditions that both
select for the replacement genome, e.g. via antibiotic resistance
markers, and select against the host genome, e.g. inserting an
inducible SacB, or like gene, in the host genome. In still another
aspect, in certain selections of host and donor genomes,
restriction endonucleases are available that cleave host genome DNA
but not donor genome DNA. In such circumstances, providing an
inducible gene that expresses such a restriction endonuclease may
be provided to remove a host genome. For example, the restriction
enzyme Fsel cleaves at four sites in E. coli, but none in H.
influenzae.
Mutant LoxP Sites for Serial Site-Specific Recombination
[0040] In one aspect, pairs of loxP sites may be used for
assembling replacement genomes or large DNA circles in accordance
with the invention. As illustrated in FIG. 4, loxP sites comprise a
left end (400), i.e. "LE" Cre recognition site, or "arm," a right
end (404), i.e. "RE," Cre recognition site, or "arm," and sandwich
between the LE and RE arms, a spacer region (402). In most wild
type and mutant loxP sites, the LE and RE arms (400 and 404) are
each 13 basepair in length, and the spacer region (402) is 8
basepairs in length. Also, in the wild type and in most mutant loxP
sites, the LE and RE arms are inverted repeats. The components of
the loxP site may be modified to produce sets of mutant loxP pairs,
as illustrated in FIG. 4, which have the following properties: (i)
members of a pair react with each other (i.e. to form
recombinants), but essentially do not react with other member pairs
of the set, and (ii) the product of a reaction between members of a
pair are one inoperable loxP site (i.e., Cre is substantially
unable to catalyze a recombination involving the site) one active
loxP site (i.e., Cre is able to catalyze a recombination involving
the site). In one aspect, the latter active loxP site is the wild
type loxP site. Such pairs of loxP sites operate as illustrated in
FIG. 4. There single mutant loxP site (408) recombines with single
mutant loxP site (410) to produce recombinant (420) that has double
mutant loxP site (422) and mutant-free loxP site (424). Single
mutant loxP site (408) comprises mutant LE (400), wild type RE
(402), and spacer region (402). Single mutant loxP site (410)
comprises wild type LE (414), mutant RE (416), and spacer region
(418). Spacer regions (402) and (418) usually (but not necessarily)
have the same sequence within a pair of interacting (or compatible)
sites. In one aspect, non-interacting loxP sites have spacer
regions with different sequences. A Cre catalyzed recombination of
mutant loxP sites (408) and (410) produces (425) a product (420)
containing two separate loxP sites in which both mutant arms are
brought together and both wild type arms are brought together.
Mutant loxP sites are selected so that whenever a double mutant
loxP is produced it is substantially inoperable with respect to
further Cre catalyzed recombinations. This prevents undesired
recombinations involving the sites when Cre is used in later steps
of serial site-specific recombination. In another aspect, the
second loxP site of recombinant (420) (which is usually the wild
type loxP site) is fully active with other compatible loxP sites
(e.g. that have the same spacer region). Thus, such sites may be
used to add further segments to a replacement genome or pairs of
such sites may be used to exchange fragments of a replacement
genome, e.g. in a recombinase mediated cassette exchange (RMCE)
type of reaction, Seibler and Bode, Biochemistry, 36: 1740-1747
(1997); and Bode et al, U.S. Pat. No. 6,992,235; which references
are incorporated by reference.
[0041] Many mutant loxP sites are available for use with the
invention. For example, six mutant spacer sites that may be used
with the invention have been described in the literature. e. g.
Nucleic Acids Res. 14, 2287-2300 (1986)); Gene 216, 55-65 (1998);
Nucleic Acids Res. 30, 3067-3077 (2002), and U.S. Pat. No.
6,465,254, which are incorporated by reference. Additional loxP
mutants may be obtained by various screening methods, e.g. as
disclosed in Missirlis et al. (cited above); Langer et al, Nucleic
Acids Research, 30: 3067-3077 (2002), and the like, which are
incorporated by reference. Table 1 lists published loxP sites. The
following formula provides a general description of pairs of loxP
sites that may be used in the invention, e.g. as illustrated in
FIGS. 1A-1E, wherein a first member of a pair is defined as:
LE.sub.1-S.sub.1-RE, and a second member of the pair is defined as:
LE.sub.2-S.sub.2-RE.sub.2 where:
[0042] LE.sub.1 is a mutant or wild type left end loxP site Cre
recognition sequence and RE, is a mutant or wild type right end
loxP site Cre recognition sequence such that whenever LE.sub.1 is a
wild type sequence, RE, is a mutant sequence, and whenever LE.sub.1
is a mutant sequence, RE.sub.1 is a wild type sequence;
[0043] LE.sub.2 is a mutant or wild type left end loxP site Cre
recognition sequence and RE.sub.2 is a mutant or wild type right
end loxP site Cre recognition sequence such that whenever LE.sub.2
is a wild type sequence, RE.sub.2 is a mutant sequence, and
whenever LE.sub.2 is a mutant sequence, RE.sub.2 is a wild type
sequence; with the proviso that whenever LE1 is a mutant sequence,
then LE2 is a wild type sequence; and
[0044] S.sub.1 and S.sub.2 are compatible non-promiscuous loxP
spacer regions. As used herein, "non-promiscuous" in reference to a
loxP spacer sequence means that loxP sites containing such sequence
(or pair of non-self recombining sequences) are substantially
unreactive, or non-cross-reactive, with loxP sites containing other
spacer sequences. In one aspect, non-promiscuous means that such
sequence or pairs of sequences cross-react with less than 100 other
loxP sites having a spacer selected from the set defined by formula
NNNTANNN; in another aspect, such cross-reactivity is with less
than 50 of such sites; in another aspect, such cross-reactivity is
with less than of 20 such sites; and in another aspect, such
cross-reactivity is with less than of 10 such sites.
[0045] In one aspect, LE.sub.1 is the lox71 left end loxP site Cre
recognition sequence whenever it is a mutant sequence and RE.sub.1
is the lox66 right end loxP site Cre recognition sequence whenever
it is a mutant sequence. Likewise, LE.sub.2 is the lox71 left end
loxP site Cre recognition sequence whenever it is a mutant sequence
and RE.sub.2 is the lox66 right end loxP site Cre recognition
sequence whenever it is a mutant sequence.
[0046] In another aspect, S.sub.1 and S.sub.2 are both the same
sequence selected from the group consisting of: TABLE-US-00001
GTATAGTA GCGTATGT GGTTACGG GGCTATAG TTGTATGG TTTTAGGT TCGTAGGC
GGATAGTA GAGTACGC GTGTATTT AGGTATGC
[0047] In still another aspect, S.sub.1 and S.sub.2 are both the
same sequence selected from the group consisting of: TABLE-US-00002
GTATAGTA GCGTATGT GGTTACGG GGCTATAG TTGTATGG TTTTAGGT TCGTAGGC
GGATAGTA GAGTACGC GTGTATTT AGGTATGC
[0048] In another aspect, S.sub.1 is GTGTACGC whenever S.sub.2 is
GTGTACGG; and S.sub.2 is GTGTACGC whenever S.sub.1 is GTGTACGG.
TABLE-US-00003 TABLE I (deviations from wild type shown in lower
case) SEQ ID Site Name LE spacer RE NO wild type ATAACTTCGTATA
ATGTATGC TATACGAAGTTAT 1 lox511 ATAACTTCGTATA ATGTATaC
TATACGAAGTTAT 2 lox5171 ATAACTTCGTATA ATGTgTaC TATACGAAGTTAT 3
lox2272 ATAACTTCGTATA AaGTATcC TATACGAAGTTAT 4 m2 ATAACTTCGTATA
AgaaAcca TATACGAAGTTAT 5 m3 ATAACTTCGTATA taaTAcca TATACGAAGTTAT 6
m7 ATAACTTCGTATA AgaTAgaa TATACGAAGTTAT 7 m11 ATAACTTCGTATA
cgaTAcca TATACGAAGTTAT 8 lox71 taccgTTCGTATA ATGTATGC TATACGAAGTTAT
9 lox66 ATAACTTCGTATA ATGTATGC TATACGAAcggta 10
[0049] FIG. 3 is a genetic map of a representative pLOX vector that
may be used for maintaining a plurality of segments for assembly
into a replacement genome. Each vector has mutant loxP site for
integration with a precursor replacement genome, and a recipient
lox P site for receiving the next incoming clone in a subsequent
assembly step. The replicon region of the vector is removed by FseI
digestion prior to transformation.
Kits of the Invention
[0050] In one aspect, kits of the invention comprise a plurality of
vectors for accepting segments as inserts, each vector comprising
at least one recombination element. Vectors for use with methods of
the invention may each further include one or more selectable
markers for determining the presence of a recombinant molecule.
Kits of the invention may further include one or more recombinases
to catalyze recombination reactions involving recombination
elements in the vectors of the kits, as well as ancillary proteins,
co-factors, and necessary buffers and salts for conducting
recombination reactions. In one embodiment, kits of the invention
include at least one Cre recombinase. Kits of the invention may
further include reagents for selecting host organisms carrying
desired recombinant molecules, including reagents for positive
and/or negative selection. Kits of the invention also include any
delivery system for delivering materials or reagents for carrying
out a method of the invention. In the context of recombination
reactions for assembling a nucleic acid construct, such delivery
systems include systems that allow for the storage, transport, or
delivery of reaction reagents (e.g., vectors, enzymes, etc. in the
appropriate containers) and/or supporting materials (e.g., buffers,
written instructions for performing the reactions etc.) from one
location to another. For example, kits include one or more
enclosures (e.g., boxes) containing the relevant reaction reagents
and/or supporting materials. Such contents may be delivered to the
intended recipient together or separately. For example, a first
container may contain an enzyme for use in a reaction, while a
second container contains vectors.
EXAMPLE I
Use of H. influenzae as Genome Donor to an E. coli Host
[0051] In this example, H. influenzae was selected as a donor
organism because of its free living status and its relatively
compact genome (1.83 Mbp). The strain of H. influenzae employed is
Rd KW20, which is kanamycin resistant and RecA negative (to
eliminate the possibility of confounding homologous recombination).
E. coli was selected as the host organism, specifically the HMS174
strain (Novagen), as it has K12 background, supports IPTG inducible
recombinant protein expression, and is RecA negative. H. influenzae
and E. coli are closely related, commensal gammaproteobacteria and
the complete genome sequence is available for both organisms
[(Science 269, 496-512 (1995); Science 277, 1453-1452 (1997)].
[0052] A BAC library was constructed from an MboI partial digest of
H. influenzae gDNA. BAC clones from this library were end sequenced
at high redundancy (>200.times. clone coverage) and mapped to
the reference H. influenzea genome sequence. A minimal tiling path
of 19 BAC clones that represents 95% of the H. influenzae genome
and 98.6% of H. influenzae genes in intact predicted operons were
selected. The tiling set was selected to maximize genome coverage,
to minimize clone overlap, and to disrupt the fewest number of
genes and predicted operons. This set of donor clones (i.e.
segments) are used for re-assembly in the host organism. A 10 kb
plasmid library was also constructed and end sequenced leading to
the selection of 15 plasmids that can close most gaps in the BAC
tiling path, if necessary.
[0053] Assays were carried out to assess the following: (i) H.
influenzea genes that cannot be cloned in E. coli. There are at
least 24 annotated genes present in the physical gaps that remain
between clones in the minimal tiling set. (Generally, different
minimal tiling sets may have different numbers of such genes).
While there is no obvious pattern in this gene set of the presently
selected minimal tiling set, there are several genes involved in
galactose metabolism and also several uncharacterized genes, which
may be dispensable. There are, however, five genes that encode what
seem to be essential proteins, including three ribosomal proteins,
a GTP-binding protein, and a DNA polymerase III subunit. These
genes appear in the predicted set of 206 essential genes from a
recent meta-analysis of all experimental and bioinformatic
approaches undertaken to date that have attempted to define the
core set of essential genes in a free living organism (Gil et al,
cited above). While the non-essential toxic genes can likely be
ignored, those that are toxic but essential are included in the
replacement genome. Such genes may be of utility in resolving the
donor and host genomes in the final hybrid organism. (ii) Which
cloned H. influenzae genes are expressed in E. coli. A primary cDNA
library was constructed from one of the BAC clones (M01) in the
minimal tiling set which contained 135 predicted genes. A total of
9,216 ESTs were sequenced and a total of 123 unambiguous hits to
Haemophilus genes were present in BAC M01. These ESTs collapsed
into 17 clusters (i.e. 17 genes were represented). Thus, despite
the limitations of this sampling procedure where most hits were
against the host cell (E. coli) transcripts, it is clear that genes
encoded in Haemophilus BACs are transcribed by the host cell
machinery. A functional expression test was performed next.
Surprisingly, the Haemophilus restriction systems appeared to be
clonable in E. coli. This may occur due to the fact that
restriction endonuclease genes are co-transcribed with a methylase
gene that protects the cell from self-digestion. The HindIII
restriction enzyme system is encoded by clone C09 in the minimal
tiling set, and it was verified that genomic DNA extracted from
this E. coli clone is in fact protected by expression of the
HindIII methylase such that it is resistant to digestion by the
HindIII restriction endonuclease.
EXAMPLE II
In Vivo Assembly of Episomal Elements in E. coli
[0054] In this example episomal elements that contain mutant LoxP
sites are constructed using standard molecular biology procedures.
These constructs are transformed sequentially into an E. coli host
and fusion is mediated by induction of Cre expression within the
host cell. Separately, Bacterial Artificial Chromosomes (BACs)
containing large segments of the H. influenzae genome are retrofit
with mutant lox sites and selectable markers using the RED/ET
system. BACs retrofitted in this manner are suitable for serial
recombination in Cre expressing RED/ET E. coli host cells.
[0055] A short DNA segment with an EcoRI compatible overhang on one
end plus a HindIII compatible overhang on the opposite end, and
containing a LoxP site that has both an LE arm mutant (ATAAC to
TACCG) and a spacer mutant (C to G at spacer position 2 and A to C
at spacer position 7) was ligated into EcoRI/HindIII cut and gel
purified pET19b expression vector. This ampicillin resistant pET19b
construct contains the coding sequence for the Cre enzyme inserted
into the NdeI site of the multiple cloning region, under control of
an IPTG inducible promoter. This construct was transformed into
HMS.sub.--174 E. coli cells (Novagen), and a batch of
electro-competent cells containing the pET19b construct was
prepared. Separately, a complementary mutant LoxP site was designed
that contained the same mutant spacer region as the above site, and
a right element arm mutation (GTTAT to CGGTA). This site was
inserted into the tetA (tetracycline resistance) gene in plasmid
pBR322 by fusion PCR, using end primers tailed with SacI and NotI
restriction sites. The PCR product, comprised of the tetA gene with
the embedded RE mutant loxP site was digested with SacI and Not and
gel purified, then ligated into SacI/NotI digested and gel purified
BAC vector pECBACl. This construct was propagated in E. coli DH10B
cells and DNA was isolated by alkaline lysis and transformed into
the electro-competent HMS.sub.--174 E. coli host cells already
harboring the pET19b LE mutant LoxP construct. Cells were grown
first on solid media containing IPTG and ampicillin, then harvested
and transferred to plates containing tetracycline and fusaric acid.
Fusaric acid is toxic to cells expressing a functional tetA gene.
Since the pECBACl construct contains a RE mutant LoxP site embedded
in the tetA gene, upon Cre mediated recombination that joins the
pECBACl and pET19b constructs, the pECBACl tetA insert is
disrupted, allowing recombinants to grow on fusaric acid containing
solid media. A total of 96 clones were screened for recombination
by colony PCR using a left primer complementary to the Cre gene in
the pET19b construct and a right primer complementary to a region
of the tetA gene in the pECBACl construct. The expected junction
fragment was observed in two of the 96 clones screened. Subsequent
sequencing of these positive clones indicated a fusion of the tetA
and Cre genes and conversion of the single arm mutant loxP sites to
a wild type site, thus verifying successful in vivo
recombination.
[0056] The RED/ET system was used to retrofit a BAC (BAC 1)
containing a large (104 kbp) segment of the H. infuenzae genome
with a mutant LoxP site. A double stranded oligonucleotide was
prepared that comprised a LoxP site with the LE arm mutation (ATAAC
to TACCG), a wild type spacer region, and EcoRI compatible overhang
on one end plus a HindIII compatible overhang on the opposite end.
This oligonucleotide was ligated into EcoRI/HindIII cut and gel
purified pET19b vector. Subsequently, a segment of this pET19b
construct containing the beta-lactamase (bla) gene for ampicillin
resistance next to the inserted LE mutant LoxP site was amplified
by PCR. The PCR primers were tailed with 44 bp sequences homologous
to a non-essential segment of the backbone of the pECBACl vector.
The H. influenzae BACl was transformed into electro-competent EL350
cells. These cells carry the Cre gene under control of an arabinose
inducible promoter, and a segment of the bacteriophage lambda
genome encoding the exo, bet and gam genes under control of a
temperature sensitive repressor (Yu et al. PNAS, 2000). Heat
induced, electro-competent EL350 cells carrying the 104 kbp H.
influenzae segment in chloramphenicol-resistant pECBACl were
prepared and transformed with the PCR product containing the bla
gene, mutant lox site and pECBACl homology arms. Transformed cells
were plated on solid media containing chloramphenicol and
ampicillin. Numerous colonies were picked and targeted insertion of
the mutant LoxP cassette was sequence verified.
[0057] For H. influenzae precursor genome assembly, the neighboring
clone in the H. influenzae BAC minimal tiling path (BAC II) is
retrofit with a cassette containing a complementary LoxP site. A
double stranded oligonucleotide is prepared that comprises a LoxP
site with the RE arm mutant (GTTAT to CGGTA), a wild type spacer
region, and SpeI compatible overhangs. This oligonucleotide is
ligated into Spe1 cut and gel purified pGPS1.1 vector.
Subsequently, a segment of this pGPS1.1 construct containing the
kanamycin resistance gene next to the inserted RE mutant LoxP site
is amplified by PCR. The PCR primers are tailed with the 44 bp
sequences, as above, that are homologous to a non-essential segment
of the backbone of the pECBACl vector. BACII is transformed into
electro-competent EL350 cells and heat induced, electro-competent
EL350 cells carrying the H. influenzae genome segment in
chloramphenicol-resistant pECBACl are prepared, followed by
transformation of the PCR product containing the kanamycing
resistance gene, RE mutant lox site and pECBAC homology arms and
integration of this cassette into BACII.
[0058] Cells containing RE loxP retrofit BACII are grown and BAC
DNA is isolated by standard alkaline lysis procedures and purified
by pulsed field gel electrophoresis (PFGE). Electro-competent EL350
cells containing LE loxP retrofitted BACI are prepared, and
transformed with purified retrofitted-BACII DNA and grown in liquid
media in the presence of L(+)-arabinose for induction of Cre gene
expression plus double antibiotics (ampicillin and kanamycin) which
are necessary to maintain both retrofit-BACI and retrofit-BACII in
the host cells to allow their recombination. The F replicon in the
BAC vector maintains stringent copy number control such that only
one or two copies are present per cell. Cells are plated and
colonies picked and screened for the presence of the BACI/BACII
fusion, which represents the first recombinant precursor genome in
the modular genome construction process. The orientation of the two
genome segments in the consolidated BAC is predetermined by
defining the orientation of the spacer regions of the LoxP sites
introduced into the original BACs, BACI and BACII. Post
recombination, the consolidated circular DNA molecule (BACI/II)
will have one reactive wild type LoxP site, an inert double arm
mutant LoxP site, an amplicillin resistance gene, a kanamycin
resistance gene and two juxtaposed copies of the pECBACl vector
backbone. Using RED/ET recombination, the wild type LoxP site and
the adjacent antibiotic resistance marker are replaced with a new
cassette that contains a new LE arm mutant loxP site and a
previously unused antibiotic resistance marker, such as the zeocin
resistance gene or the gentamycin resistance gene. The next
adjacent clone in the tiling path (BACIII) is retrofit as above
with a complementary RE arm mutant site and an antibiotic
resistance gene, which may be the antibiotic resistance gene
removed from BACI/II or a different antibiotic resistance gene. DNA
is prepared from BACIII and transformed into host cells harboring
BACI/II. Cells are grown in liquid media in the presence of
L(+)-arabinose for induction of Cre gene expression and the
appropriate double antibiotics to maintain both BACI/II and BACIII
in the host cells, so as to allow their recombination. In this
manner, each BAC in the tiling path is added to the growing
precursor genome until the genome is tiling path is completely
reassembled, or until a desired state of re-assembly is
achieved.
Definitions
[0059] Terms and symbols of nucleic acid chemistry, biochemistry,
genetics, and molecular biology used herein follow those of
standard treatises and texts in the field, e.g. Kornberg and Baker,
DNA Replication, Second Edition (W.H. Freeman, New York, 1992);
Lehninger, Biochemistry, Second Edition (Worth Publishers, New
York, 1975); Strachan and Read, Human Molecular Genetics, Second
Edition (Wiley-Liss, New York, 1999); Eckstein, editor,
Oligonucleotides and Analogs: A Practical Approach (Oxford
University Press, New York, 1991); Gait, editor, Oligonucleotide
Synthesis: A Practical Approach (IRL Press, Oxford, 1984); and the
like.
[0060] "Amplicon" means the product of a polynucleotide
amplification reaction. That is, it is a population of
polynucleotides, usually double stranded, that are replicated from
one or more starting sequences. The one or more starting sequences
may be one or more copies of the same sequence, or it may be a
mixture of different sequences. Amplicons may be produced by a
variety of amplification reactions whose products are multiple
replicates of one or more target nucleic acids. Generally,
amplification reactions producing amplicons are "template-driven"
in that base pairing of reactants, either nucleotides or
oligonucleotides, have complements in a template polynucleotide
that are required for the creation of reaction products. In one
aspect, template-driven reactions are primer extensions with a
nucleic acid polymerase or oligonucleotide ligations with a nucleic
acid ligase. Such reactions include, but are not limited to,
polymerase chain reactions (PCRs), linear polymerase reactions,
nucleic acid sequence-based amplification (NASBAs), rolling circle
amplifications, and the like, disclosed in the following references
that are incorporated herein by reference: Mullis et al, U.S. Pat.
Nos. 4,683,195; 4,965,188; 4,683,202; 4,800,159 (PCR); Gelfand et
al, U.S. Pat. No. 5,210,015 (real-time PCR with "taqman" probes);
Wittwer et al, U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No.
5,399,491 ("NASBA"); Lizardi, U.S. Pat. No. 5,854,033; Aono et al,
Japanese patent pubi. JP 4-262799 (rolling circle amplification);
and the like. In one aspect, amplicons of the invention are
produced by PCRs. An amplification reaction may be a "real-time"
amplification if a detection chemistry is available that permits a
reaction product to be measured as the amplification reaction
progresses, e.g. "real-time PCR" described below, or "real-time
NASBA" as described in Leone et al, Nucleic Acids Research, 26:
2150-2155 (1998), and like references. As used herein, the term
"amplifying" means performing an amplification reaction. A
"reaction mixture" means a solution containing all the necessary
reactants for performing a reaction, which may include, but not be
limited to, buffering agents to maintain pH at a selected level
during a reaction, salts, co-factors, scavengers, and the like.
[0061] "Duplex" means at least two oligonucleotides and/or
polynucleotides that are fully or partially complementary undergo
Watson-Crick type base pairing among all or most of their
nucleotides so that a stable complex is formed. The terms
"annealing" and "hybridization" are used interchangeably to mean
the formation of a stable duplex. In one aspect, stable duplex
means that a duplex structure is not destroyed by a stringent wash,
e.g. conditions including temperature of about 5.degree. C. less
that the T.sub.m of a strand of the duplex and low monovalent salt
concentration, e.g. less than 0.2 M, or less than 0.1 M. "Perfectly
matched" in reference to a duplex means that the poly- or
oligonucleotide strands making up the duplex form a double stranded
structure with one another such that every nucleotide in each
strand undergoes Watson-Crick basepairing with a nucleotide in the
other strand. The term "duplex" comprehends the pairing of
nucleoside analogs, such as deoxyinosine, nucleosides with
2-aminopurine bases, PNAs, and the like, that may be employed. A
"mismatch" in a duplex between two oligonucleotides or
polynucleotides means that a pair of nucleotides in the duplex
fails to undergo Watson-Crick bonding.
[0062] "Hybridization" refers to the process in which two
single-stranded polynucleotides bind non-covalently to form a
stable double-stranded polynucleotide. The term "hybridization" may
also refer to triple-stranded hybridization. The resulting
(usually) double-stranded polynucleotide is a "hybrid" or "duplex."
"Hybridization conditions" will typically include salt
concentrations of less than about 1M, more usually less than about
500 mM and less than about 200 mM. Hybridization temperatures can
be as low as 5.degree. C., but are typically greater than
22.degree. C., more typically greater than about 30.degree. C., and
preferably in excess of about 37.degree. C. Hybridizations are
usually performed under stringent conditions, i.e. conditions under
which a probe will hybridize to its target subsequence. Stringent
conditions are sequence-dependent and are different in different
circumstances. Longer fragments may require higher hybridization
temperatures for specific hybridization. As other factors may
affect the stringency of hybridization, including base composition
and length of the complementary strands, presence of organic
solvents and extent of base mismatching, the combination of
parameters is more important than the absolute measure of any one
alone. Generally, stringent conditions are selected to be about
5.degree. C. lower than the T.sub.m for the specific sequence at s
defined ionic strength and pH. Exemplary stringent conditions
include salt concentration of at least 0.01 M to no more than 1 M
Na ion concentration (or other salts) at a pH 7.0 to 8.3 and a
temperature of at least 25.degree. C. For example, conditions of
5.times.SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4)
and a temperature of 25-30.degree. C. are suitable for
allele-specific probe hybridizations. For stringent conditions, see
for example, Sambrook, Fritsche and Maniatis. "Molecular Cloning A
laboratory Manual" 2.sup.nd Ed. Cold Spring Harbor Press (1989) and
Anderson "Nucleic Acid Hybridization" 1.sup.st Ed., BIOS Scientific
Publishers Limited (1999), which are hereby incorporated by
reference in its entirety for all purposes above. "Hybridizing
specifically to" or "specifically hybridizing to" or like
expressions refer to the binding, duplexing, or hybridizing of a
molecule substantially to or only to a particular nucleotide
sequence or sequences under stringent conditions when that sequence
is present in a complex mixture (e.g., total cellular) DNA or
RNA.
[0063] "Inducible" or "inducible control" in reference to gene
expression means that gene expression is controlled by a promoter
and possibly of regulatory elements such that a promoter is
transcriptionally active under a specific set of conditions, e.g.,
a change in physical conditions, such as a change in pH,
temperature, salt concentration, or the like, or the presence of a
particular chemical signal or combination of chemical signals that,
for example, affect binding of the transcriptional activator to the
promoter and/or affect function of the transcriptional activator
itself.
[0064] "Ligation" means to form a covalent bond or linkage between
the termini of two or more nucleic acids, e.g. oligonucleotides
and/or polynucleotides, in a template-driven reaction. The nature
of the bond or linkage may vary widely and the ligation may be
carried out enzymatically or chemically. As used herein, ligations
are usually carried out enzymatically to form a phosphodiester
linkage between a 5' carbon of a terminal nucleotide of one
oligonucleotide with 3' carbon of another oligonucleotide. A
variety of template-driven ligation reactions are described in the
following references, which are incorporated by reference: Whitely
et al, U.S. Pat. No. 4,883,750; Letsinger et al, U.S. Pat. No.
5,476,930; Fung et al, U.S. Pat. No. 5,593,826; Kool, U.S. Pat. No.
5,426,180; Landegren et al, U.S. Pat. No. 5,871,921; Xu and Kool,
Nucleic Acids Research, 27: 875-881 (1999); Higgins et al, Methods
in Enzymology, 68: 50-71 (1979); Engler et al, The Enzymes, 15:
3-29(1982); and Namsaraev, U.S. patent publication
2004/0110213.
[0065] "Nucleic acid construct" is used synonymously with
"recombinant DNA molecule."
[0066] "Nucleoside" as used herein includes the natural
nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g. as
described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman,
San Francisco, 1992). "Analogs" in reference to nucleosides
includes synthetic nucleosides having modified base moieties and/or
modified sugar moieties, e.g. described by Scheit, Nucleotide
Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical
Reviews, 90: 543-584 (1990), or the like, with the proviso that
they are capable of specific hybridization. Such analogs include
synthetic nucleosides designed to enhance binding properties,
reduce complexity, increase specificity, and the like.
Polynucleotides comprising analogs with enhanced hybridization or
nuclease resistance properties are described in Uhlman and Peyman
(cited above); Crooke et al, Exp. Opin. Ther. Patents, 6: 855-870
(1996); Mesmaeker et al, Current Opinion in Structual Biology, 5:
343-355 (1995); and the like. Exemplary types of polynucleotides
that are capable of enhancing duplex stability include
oligonucleotide N3.fwdarw.P5' phosphoramidates (referred to herein
as "amidates"), peptide nucleic acids (referred to herein as
"PNAs"), oligo-2'-O-alkylribonucleotides, polynucleotides
containing C-5 propynylpyrimidines, locked nucleic acids (LNAs),
and like compounds. Such oligonucleotides are either available
commercially or may be synthesized using methods described in the
literature.
[0067] "Polymerase chain reaction," or "PCR," means a reaction for
the in vitro amplification of specific DNA sequences by the
simultaneous primer extension of complementary strands of DNA. In
other words, PCR is a reaction for making multiple copies or
replicates of a target nucleic acid flanked by primer binding
sites, such reaction comprising one or more repetitions of the
following steps: (i) denaturing the target nucleic acid, (ii)
annealing primers to the primer binding sites, and (iii) extending
the primers by a nucleic acid polymerase in the presence of
nucleoside triphosphates. Usually, the reaction is cycled through
different temperatures optimized for each step in a thermal cycler
instrument. Particular temperatures, durations at each step, and
rates of change between steps depend on many factors well-known to
those of ordinary skill in the art, e.g. exemplified by the
references: McPherson et al, editors, PCR: A Practical Approach and
PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995,
respectively). For example, in a conventional PCR using Taq DNA
polymerase, a double stranded target nucleic acid may be denatured
at a temperature >90.degree. C., primers annealed at a
temperature in the range 50-75.degree. C., and primers extended at
a temperature in the range 72-78.degree. C. Reaction volumes
typically range from a few hundred nanoliters, e.g. 200 mL, to a
few hundred .mu.L, e.g. 200 .mu.L.
[0068] "Polynucleotide" or "oligonucleotide" are used
interchangeably and each mean a linear polymer of nucleotide
monomers. Monomers making up polynucleotides and oligonucleotides
are capable of specifically binding to a natural polynucleotide by
way of a regular pattern of monomer-to-monomer interactions, such
as Watson-Crick type of base pairing, base stacking, Hoogsteen or
reverse Hoogsteen types of base pairing, or the like. Such monomers
and their internucleosidic linkages may be naturally occurring or
may be analogs thereof, e.g. naturally occurring or non-naturally
occurring analogs. Non-naturally occurring analogs may include
PNAs, phosphorothioate internucleosidic linkages, bases containing
linking groups permitting the attachment of labels, such as
fluorophores, or haptens, and the like. Whenever the use of an
oligonucleotide or polynucleotide requires enzymatic processing,
such as extension by a polymerase, ligation by a ligase, or the
like, one of ordinary skill would understand that oligonucleotides
or polynucleotides in those instances would not contain certain
analogs of internucleosidic linkages, sugar moities, or bases at
any or some positions. Polynucleotides typically range in size from
a few monomeric units, e.g. 5-40, when they are usually referred to
as "oligonucleotides," to several thousand monomeric units.
Whenever a polynucleotide or oligonucleotide is represented by a
sequence of letters (upper or lower case), such as "ATGCCTG," it
will be understood that the nucleotides are in 5.fwdarw.3' order
from left to right and that "A" denotes deoxyadenosine, "C" denotes
deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes
thymidine, "I" denotes deoxyinosine, "U" denotes uridine, unless
otherwise indicated or obvious from context. Unless otherwise noted
the terminology and atom numbering conventions will follow those
disclosed in Strachan and Read, Human Molecular Genetics 2
(Wiley-Liss, New York, 1999). Usually polynucleotides comprise the
four natural nucleosides (e.g. deoxyadenosine, deoxycytidine,
deoxyguanosine, deoxythymidine for DNA or their ribose counterparts
for RNA) linked by phosphodiester linkages; however, they may also
comprise non-natural nucleotide analogs, e.g. including modified
bases, sugars, or internucleosidic linkages. It is clear to those
skilled in the art that where an enzyme has specific
oligonucleotide or polynucleotide substrate requirements for
activity, e.g. single stranded DNA, RNA/DNA duplex, or the like,
then selection of appropriate composition for the oligonucleotide
or polynucleotide substrates is well within the knowledge of one of
ordinary skill, especially with guidance from treatises, such as
Sambrook et al, Molecular Cloning, Second Edition (Cold Spring
Harbor, Laboratory, New York, 1989), and like references.
[0069] "Primer" means an oligonucleotide, either natural or
synthetic, that is capable, upon forming a duplex with a
polynucleotide template, of acting as a point of initiation of
nucleic acid synthesis and being extended from its 3' end along the
template so that an extended duplex is formed. The sequence of
nucleotides added during the extension process are determined by
the sequence of the template polynucleotide. Usually primers are
extended by a DNA polymerase. Primers usually have a length in the
range of from 14 to 36 nucleotides.
[0070] "Recombination element" means a sequence that is a site of
recombination of DNA sequences in a recombination reaction. A
recombination element may be a segment of DNA that is homologous to
another segment that participates in a recombination reaction (e.g.
as in homologous recombination), or it may be a specific sequence
where recombination takes place by action of an associated
recombinase, and perhaps additional ancillary factors, that
recognizes all or part of the specific sequence (e.g. as in
site-specific recombination). In one aspect, a recombination
element is a recombination site of a site-specific recombination
system, such as Cre-LoxP, Flp-FRT, or the like.
[0071] "Regulatory elements" in reference to gene expression means
DNA sequences that are operably linked to the expression of one or
more genes. Such elements are commonly located at positions
adjacent to the expressed genes and can include promoters,
terminators, antiterminators, activators, attenuators, and the
like, e.g. Kornberg and Baker, DNA Replication, 2.sup.nd Edition
(Freeman, San Francisco, 1992), Makrides, Microbiological Reviews,
60: 512-538 (1996). Frequently, one or more co-regulated genes are
associated with the same set of regulatory elements in an
operon.
[0072] "Synthetic" in reference to a polynucleotide segment of the
invention means that all or a portion of the segment is constructed
from one or more polynucleotides that were initially chemically
synthesized. After synthesis, such synthetic polynucleotide
segments may be replicated by in vivo or in vitro enzymatic
methods, e.g. by conventional cloning or by amplification, such as
by PCR, RCR, or the like. Various approaches may be used to
constructing synthetic polynucleotide segments for use with the
invention including, but not limited to, those described in the
following references that are incorporated herein by reference:
Tian et al, Nature, 432: 1050-1054 (2004); Soldatov et al,
International patent publication WO 2004/092375; U.S. patent
publication 2003/0138782A1; U.S. patent publication 2003/0165946A1;
U.S. patent publication 2005/0106606A 1; International patent
publication WO 2006/044956; Cleary et al, Nature Methods, 1:
241-248 (2004); Zhou et al, Nucleic Acids Research, 32: 5409-5417
(2004); Chen et al, J. Chem. Soc., 116: 8799-8800 (1994); Mandecki
et al, Gene, 68: 101-107 (1988); Kodumal et al, Proc. Natl. Acad.
Sci., 101: 15573-15578 (2004); Smith et al, Proc. Natl. Acad. Sci.,
100: 15440-15445 (2003); or the like.
Sequence CWU 1
1
10 1 34 DNA Escherichia coli 1 ataacttcgt ataatgtatg ctatacgaag
ttat 34 2 34 DNA Escherichia coli 2 ataacttcgt ataatgtata
ctatacgaag ttat 34 3 34 DNA Escherichia coli 3 ataacttcgt
ataatgtgta ctatacgaag ttat 34 4 34 DNA Escherichia coli 4
ataacttcgt ataaagtatc ctatacgaag ttat 34 5 34 DNA Escherichia coli
5 ataacttcgt ataagaaacc atatacgaag ttat 34 6 34 DNA Escherichia
coli 6 ataacttcgt atataatacc atatacgaag ttat 34 7 34 DNA
Escherichia coli 7 ataacttcgt ataagataga atatacgaag ttat 34 8 34
DNA Escherichia coli 8 ataacttcgt atacgatacc atatacgaag ttat 34 9
34 DNA Escherichia coli 9 taccgttcgt ataatgtatg ctatacgaag ttat 34
10 34 DNA Escherichia coli 10 ataacttcgt ataatgtatg ctatacgaac ggta
34
* * * * *