U.S. patent application number 16/056343 was filed with the patent office on 2018-11-29 for method of nucleic acid cassette assembly.
This patent application is currently assigned to SYNTHETIC GENOMICS, INC.. The applicant listed for this patent is Synthetic Genomics, Inc.. Invention is credited to Daniel G. Gibson, Clyde A. Hutchison, III, Hamilton O. Smith, J. Craig Venter.
Application Number | 20180340165 16/056343 |
Document ID | / |
Family ID | 39107256 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340165 |
Kind Code |
A1 |
Venter; J. Craig ; et
al. |
November 29, 2018 |
METHOD OF NUCLEIC ACID CASSETTE ASSEMBLY
Abstract
Methods are provided for constructing a synthetic genome,
comprising generating and assembling nucleic acid cassettes
comprising portions of the genome, wherein at least one of the
nucleic acid cassettes is constructed from nucleic acid components
that have been chemically synthesized, or from copies of the
chemically synthesized nucleic acid components. In one embodiment,
the entire synthetic genome is constructed from nucleic acid
components that have been chemically synthesized, or from copies of
the chemically synthesized nucleic acid components. Synthetic
genomes or synthetic cells may be used for a variety of purposes,
including the generation of synthetic fuels, such as hydrogen or
ethanol.
Inventors: |
Venter; J. Craig; (La Jolla,
CA) ; Smith; Hamilton O.; (San Diego, CA) ;
Hutchison, III; Clyde A.; (La Jolla, CA) ; Gibson;
Daniel G.; (Carlsbad, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Synthetic Genomics, Inc. |
La Jolla |
CA |
US |
|
|
Assignee: |
SYNTHETIC GENOMICS, INC.
|
Family ID: |
39107256 |
Appl. No.: |
16/056343 |
Filed: |
August 6, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11635355 |
Dec 6, 2006 |
10041060 |
|
|
16056343 |
|
|
|
|
60742542 |
Dec 6, 2005 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/10 20130101;
C12N 15/1093 20130101; C12N 15/66 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12N 15/66 20060101 C12N015/66 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
No. DE-FG02-02ER63453 awarded by the Department of Energy. The
government has certain rights in the invention.
Claims
1. A method for assembling a nucleic acid construct comprising: (a)
generating a plurality of double-stranded nucleic acid cassettes
from chemically synthesized oligonucleotides or copies thereof,
wherein the cassettes comprise adjacent regions of a nucleic acid
construct to be assembled and wherein each of the cassettes overlap
with one or more other cassette(s) by at least 50 nucleotides and;
(b) simultaneously assembling the plurality of nucleic acid
cassettes in vitro in a chew back and repair step utilizing: an
enzyme having 3' or 5' exonuclease activity, a DNA polymerase,
polyethylene glycol (PEG) or a single-stranded binding protein, and
a ligase, thereby assembling the nucleic acid construct.
2. The method of claim 1 wherein the enzyme having exonuclease
activity is a 5' exonuclease and the step (b) utilizes PEG.
3. The method of claim 2 wherein the assembly is performed in a
single step.
4. The method of claim 2 wherein the plurality of nucleic acid
cassettes comprises more than 4 nucleic acid cassettes.
5. The method of claim 4 wherein the plurality of nucleic acid
cassettes comprises more than 6 nucleic acid cassettes.
6. The method of claim 2, wherein the cassettes are from about 4.5
kilobases to about 6.5 kilobases in length, not including
overlaps.
7. The method of claim 1, wherein the cassettes are about 5 kilo
bases in length, not including overlaps.
8. The method of claim 2, wherein each of the cassettes overlaps an
adjacent cassette by at least 200 nucleotides.
9. The method of claim 1, wherein the assembled nucleic acid
construct is a non-naturally occurring genome nucleic acid
construct.
10. The method of claim 1, wherein one or more of the cassettes
includes restriction enzyme sites or adaptor sites.
11. The method of claim 1, wherein each cassette is generated
entirely from chemically synthesized oligonucleotides or copies
thereof
12. The method of claim 2, wherein the method is automated.
13. The method of claim 1, wherein generating the cassettes
comprises ligating the chemically synthesized oligonucleotides to
form ligation products and performing cycles of polymerase cycle
assembly (PCA) on the ligation products, thereby forming PCA
products.
14. The method of claim 13, wherein the PCA consists of 5 cycles of
PCA and the ligation is performed at 50.degree. C.
15. The method of claim 1, wherein step (a) further comprises
cloning or amplifying the cassettes.
16. The method of claim 1, wherein the chemically synthesized
oligonucleotides or copies thereof are between about 30 nucleotides
and 1 kilo base in length.
17. The method of claim 1, wherein each of the cassettes overlaps
an adjacent cassette by about 50-1300 nucleotides.
18. The method of claim 1 wherein the plurality of nucleic acid
cassettes comprises at least 4 nucleic acid cassettes.
19. The method of claim 18 wherein the plurality of nucleic acid
cassettes comprises more than 6 nucleic acid cassettes.
20. The method of claim 1, wherein the cassettes are from about 4.5
kilobases to about 6.5 kilobases in length, not including
overlaps.
21. The method of claim 1, wherein each of the cassettes overlaps
an adjacent cassette by at least 200 nucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
patent application Ser. No. 11/635,355 filed on Dec. 6, 2006
entitled "Method of Nucleic Acid Cassette Assembly, now issued as
U.S. Pat. No. 10,041,060; which claims the benefit and priority
from U.S. Provisional Patent Application Ser. No. 60/742,542 filed
on Dec. 6, 2005, entitled "Synthetic Genomes." U.S. patent
application Ser. No. 11/635,355 is related to U.S. Provisional
Patent Application Ser. No. 60/752,965 filed on Dec. 23, 2005,
entitled "Introduction of Genomes into Microorganisms;" U.S.
Provisional Patent Application Ser. No. 60/741,469 filed on Dec. 2,
2005, entitled "Error Correction Method;" and U.S. Non-Provisional
patent application Ser. No. 11/502,746 filed on Aug. 11, 2006,
entitled "In Vitro Recombination Method," all of which are
incorporated herein by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB
[0003] The entire content of the following electronic submission of
the sequence listing via the USPTO EFS-Web server, as authorized
and set forth in MPEP .sctn. 1730 II.B.2(a), is incorporated herein
by reference in its entirety for all purposes. The sequence listing
is identified on the electronically filed text file as follows:
TABLE-US-00001 File Name Date of Creation Size SGI1360-2_ST25.txt
Aug. 3, 2018 1 KB
BACKGROUND OF THE INVENTION
Field of the Invention
[0004] The present invention relates generally to molecular
biology, and more particularly to synthetic genomes.
Description of Related Art
[0005] Conventional genetic engineering techniques are limited to
allowing manipulation of existing sequences. It would thus be
desirable to have the ability to implement dramatic alterations and
arrangements of genetic content, beyond that made possible by
conventional techniques. Consequently, there is a need for
synthetic genomes.
SUMMARY OF THE INVENTION
[0006] Embodiments and methods are provided for the design,
synthesis, assembly and expression of synthetic genomes. Included
are methods for rationally designing components of a genome;
generating small nucleic acid fragments and assembling them into
cassettes comprising portions of the genome; correcting errors in
the sequences of the cassettes; cloning the cassettes (e.g., by in
vitro methods such as rolling circle amplification); assembling the
cassettes to form a synthetic genome (e.g., by methods of in vitro
recombination); and transferring the synthetic genome into a
biochemical system (e.g., by transplanting it into an intact cell,
ghost cell devoid of functioning DNA, or other vesicle). In one
embodiment, the synthetic genome comprises sufficient information
to achieve replication of a vesicle (e.g., a cell) in which it
resides. The technology extends to useful end products that a
synthetic genomic system can produce, such as energy sources (e.g.,
hydrogen or ethanol), and biomolecules such as therapeutics and
industrial polymers.
[0007] Included are methods for constructing a synthetic genome,
comprising generating and assembling the nucleic acid components of
the genome, wherein at least part of the genome is constructed from
nucleic acid components that have been chemically synthesized, or
from copies of chemically synthesized nucleic acid components. In
one embodiment, an entire synthetic genome is constructed from
nucleic acid components that have been chemically synthesized, or
from copies of chemically synthesized nucleic acid components.
Further, a synthetic genome may be a synthetic cellular genome (a
genome which comprises all of the sequences required for
replication of a vesicle (e.g., a cell or synthetic vesicle) in
which it resides).
[0008] Methods are provided for constructing a synthetic cell,
comprising use of certain exemplary methods to construct a
synthetic genome and introducing (transplanting) the synthetic
genome into a vesicle (e.g., a cell or a synthetic membrane bound
vesicle). Another method includes constructing a self-replicating
synthetic cell, comprising use of exemplary methods to construct a
synthetic cellular genome and introducing (transplanting) the
synthetic cellular genome into a vesicle (e.g., a cell or a
synthetic membrane bound vesicle), under conditions effective for
the synthetic cell to replicate. Further methods include producing
a product of interest, comprising culturing an exemplary synthetic
cell under conditions effective to produce the product. When the
product is produced from a synthetic cell comprising a synthetic
cellular genome, the genome is contacted with the vesicle under
conditions effective to replicate the synthetic cell and to produce
the product.
[0009] Other exemplary methods include making a synthetic cell,
comprising removing part of or all of the resident (original)
genome from a microorganism, such as a unicellular microorganism
(e.g., a bacterium, fungus, etc.) and replacing it with a synthetic
genome that is foreign to the organism (e.g., is from a different
species of microorganism (e.g., bacterium)), which exhibits at
least one property that is different from the resident genome.
Various exemplary embodiments include a synthetic cell produced by
this method.
[0010] One exemplary embodiment includes a synthetic genome that is
capable of directing replication of a vesicle (e.g., cell) in which
it resides, under particular environmental (e.g., nutritional or
physical) conditions. In one embodiment, the cellular genome is
supplemented in the vesicle (e.g., cell) by small molecules, such
as nutrients, ATP, lipids, sugars, phosphates etc., which serve as
precursors for structural features or substrates for metabolic
functions; and/or is supplemented with complex components, such as
ribosomes, functional cell membranes, etc. These additional
elements may complement or facilitate the ability of the genome to
achieve (e.g., program) replication of the vesicle/cell. In another
embodiment, the sequences in the genome are capable of providing
all of the machinery and components required to produce a cell and
to allow the cell to replicate under particular energy or
environmental (e.g., nutritional) conditions.
[0011] Further embodiments and methods include a "minimal genome"
that may serve as a platform for introducing other sequences of
interest, such as genes encoding biologic agents (e.g., therapeutic
agents, drugs, vaccines or the like), or genes encoding products
that, in the presence of suitable precursors, can produce useful
compounds (e.g., biofuels, industrial organic chemicals, etc.). In
one embodiment, the other sequences of interest result in the
production of products in sufficient quantities to be commercially
valuable.
[0012] According to one exemplary embodiment and method, a
synthetic version of the Mycoplasma genitalium genome having 482
protein-coding genes and 43 RNA genes comprising a 580-kilobase
circular chromosome is assembled from gene cassettes. Each cassette
may be made from chemically synthesized oligonucleotides. Several
versions of each cassette may be made such that combinatorial
assembly into a complete chromosome results in millions of
different genomes. These genomes may be tested for functionality by
"genome transplantation," replacement of a cell's resident
chromosome by the synthetic genome. According to further
embodiments and methods, synthetic cells may be assembled from
various subcellular components. Additionally, a genome in a
cell-free environment may be established, comprising the necessary
transcriptional and translation "machinery" to express genes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows an illustration of suitable oligonucleotides
for preparing M. genitalium. The triangles mark the position of
transposon insertions (upper triangles are from Clyde A. Hutchison,
III, et al. Science 286, 2165 (1999)). Vertical lines delineate the
borders of the "5 kb" segments.
[0014] FIG. 2 illustrates the use of mycoplasma comparative
genomics to identify genes that may be involved in a minimal gene
set for mycoplasma. The figure displays a MUMMER comparison of the
M. capricolum and M. mycoides genomes at the protein level showing
the locations of orthologous genes. The cross pattern shows the
conservation of gene position relative to the origin and terminus
of replication for these two species.
[0015] FIG. 3A shows the design of 682 48-mer oligonucleotides, and
the assembly of those oligonucleotides into three overlapping
segments (cassettes), S1 (6,528 bp), S2 (5,328 bp), and S3 (4,508
bp), to assemble the mouse mitochondrial genome. The BstXI site in
S1 (CCAATGAAATGG; SEQ ID NO: 1), BstXI site in S2 (CCAAGTCTCTGG;
SEQ ID NO: 2), and a terminal BstXI site (CCACTGTGCTGG; SEQ ID NO:
3) are shown.
[0016] FIG. 3B displays a gel containing products from an assembly
of these pieces by the method described in Smith et al. (2003),
modified to reduce the heating damage to the synthetic product. Taq
ligations at 50.degree. C. of the oligos for each mitochondrial
fragment were subjected to 5 cycles of PCA and 20 cycles of PCR
using end primers.
[0017] FIG. 4 shows the synthesis of 5 kb cassettes of an essential
region of the M. genitalium genome.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The following descriptions of various terms as used herein
are not exhaustive and may include other descriptive matter.
[0019] "Cellular genome" or a "synthetic cellular genome" is a
genome that comprises sequences which encode and may express
nucleic acids and proteins required for some or all of the
processes of transcription, translation, energy production,
transport, production of cell membranes and components of the cell
cytoplasm, DNA replication, cell division, and the like. A
"cellular genome" differs from a viral genome or the genome of an
organelle, at least in that a cellular genome contains the
information for replication of a cell, whereas viral and organelle
genomes contain the information to replicate themselves (sometimes
with the contribution of cellular factors), but they lack the
information to replicate the cell in which they reside.
[0020] "Foreign" gene or genome is a gene or genome derived from a
source other than the resident (original) organism, e.g., from a
different species of the organism.
[0021] "Genome" may include viral genomes, the genomes of
organelles (e.g., mitochondria or chloroplasts), and genomes of
self-replicating organisms, such as bacteria, yeast, archebacteria,
or eukaryotes. A genome may also be an entirely new construct for
an organism that does not fall into any known Linnean category. In
one embodiment, the genes are from a microorganism, e.g., a
unicellular microorganism, such as a bacterium. The genes may be in
the order found in the microorganism, or they may be shuffled; and
mutant versions of some of the genes may also be included.
[0022] "Membrane-bound vesicle," refers to a vesicle in which a
lipid-based protective material encapsulates an aqueous
solution.
[0023] "Minimal genome," with respect to a cell, as used herein,
refers to a genome consisting of or consisting essentially of a
minimal set of genetic sequences that are sufficient to allow for
cell survival under specified environmental (e.g., nutritional)
conditions. A "minimal genome," with respect to an organelle, as
used herein, refers to a genome consisting of or consisting
essentially of a minimal set of genetic sequences that are
sufficient to allow the organelle to function. A minimal genome
must contain sufficient information to allow the cell or organelle
to carry out essential biological processes, such as, for example,
transcription, translation, use of an energy source, transport of
salts, nutrients and the like into and out of the organelle or
cell, etc. A "minimal replicating genome," with respect to either a
cell or an organelle, contains, in addition, genetic sequences
sufficient to allow for self replication of the cell or organelle,
Thus, a "minimal replicating synthetic genome" is a single
polynucleotide or group of polynucleotides that is at least
partially synthetic and that contains the minimal set of genetic
sequences for a cell or organelle to survive and replicate under
specific environmental conditions.
[0024] "Nucleic acid" and "Polynucleotide" are used interchangeably
herein. They include both DNA and RNA. Other types of nucleic
acids, such as PNA, LNA, modified
[0025] DNA or RNA, etc. are also included, provided that they can
participate in the synthetic operations described herein and
exhibit the desired properties and functions. A skilled worker will
recognize which forms of nucleic acid are applicable for any
particular embodiment or method described herein.
[0026] "Synthetic genome," includes a single polynucleotide or
group of polynucleotides that contain the information for a
functioning organelle or organism to survive and, optionally,
replicate itself where particular environmental (e.g., nutritional
or physical) conditions are met. All or at least part of the genome
(e.g., a cassette) is constructed from components that have been
chemically synthesized, or from copies of chemically synthesized
nucleic acid components. The copies may be produced by any of a
variety of methods, including cloning and amplification by in vivo
or in vitro methods. In one embodiment, an entire genome is
constructed from nucleic acid that has been chemically synthesized,
or from copies of chemically synthesized nucleic acid components.
Such a genome is sometimes referred to herein as a "completely
synthetic" genome. In other embodiments, one or more portions of
the genome may be assembled from naturally occurring nucleic acid,
nucleic acid that has been cloned, or the like. Such a genome is
sometimes referred to herein as a "partially synthetic" genome.
[0027] Synthetic genomes offer numerous advantages over traditional
recombinant DNA technology. For example, the selection and
construction of synthetic genome sequences allow for easier
manipulation of sequences than with classical recombination
techniques, and permits the construction of novel organisms and
biological systems. Furthermore, various embodiments and methods
are amenable to automation and adaptation to high throughput
methods, allowing for the production of synthetic genomes and
synthetic cells by computer-mediated and robotic methods that do
not require human intervention. The inventive technology opens the
door to an integrated process of synthetic genome design,
construction, introduction into a biological system, biological
production of useful products, and recursive improvement to the
design.
[0028] Various forms of rational or intelligent design of nucleic
acids may be employed according to various exemplary embodiments
and methods. According to one method, a gene set is identified that
constitutes a minimal genome, e.g., of a bacterium, such as
Mycoplasma genitalia (M. genitalium), M. capricolum (e.g.,
subspecies capricolum), E. coli, B. subtilis, or others. One or
more conventional or novel methods, or combinations thereof, may be
used to accomplish this end. One method includes using random
saturation global transposon mutagenesis to knock out the function
of each gene in a microbial genome (e.g., a bacterial genome)
individually, and to determine on this basis putative genes that
may be eliminated without destroying cell viability. See, e.g.,
Smith et al. (1999) Proc Natl Acad Sci USA 87, 826-830. Another
method is to use comparative genomics of a variety of related
genomes (e.g., analyzing the sequences of orthologous organisms,
metagenomics, etc.) to predict common genes which are basic to the
function of a microorganism of interest (e.g., a bacterium), e.g.,
to identify genes common to all members of a taxon. Existing
databases may be used, or new databases may be generated by
sequencing additional organisms, using conventional methods.
According to one method, the identification of genes in a minimal
genome is facilitated by isolating and expanding clones of
individual cells, using a method for disrupting cell aggregates.
Example I herein illustrates the use of mycoplasma comparative
genomics to identify genes that may be involved in a minimal gene
set for mycoplasma.
[0029] Following the identification of a putative minimal set of
genes required for viability and, optionally, replication under a
defined set of conditions, a candidate minimal genome may be
constructed as described herein. According to one method, a set of
overlapping nucleic acid cassettes are constructed, each generally
having about 5 kb, which comprise subsets of the genes; and the
cassettes are then assembled to form the genome. The
function/activity of the genome may be further studied by
introducing the assembled genome into a suitable biological system
and monitoring one or more functions/activities encoded by the
genome. The synthetic genome may be further manipulated, for
example, by modifying (e.g., deleting, altering individual
nucleotides, etc.) portions of genes or deleting entire genes
within one or more of the cassettes; by replacing genes or
cassettes by other genes or cassettes, such as functionally related
genes or groups of genes; by rearranging the order of the genes or
cassettes (e.g., by combinatorial assembly); etc. The consequences
of such manipulations may be examined by re-introducing the
synthetic genes into a suitable biological system. Factors that may
be considered include, e.g., growth rate, nutritional requirements
and other metabolic factors. In this manner, one may further refine
which genes are required for a minimal genome.
[0030] Another aspect of rational design according to further
methods involves the determination of which sites within a
synthetic genome may withstand insertions, such as unique
identifiers (e.g., watermarks), expressible sequences of interest,
etc., without disrupting gene function. In general, sites within a
genome that can withstand such disruption lie at the junctions
between genes, in non-coding regions, or the like.
[0031] Another aspect of rational design according to even further
methods includes the selection of suitable regulatory control
elements. For instance, in the case of prokaryotic-type cells, such
regulatory control elements include promoters, terminators, signals
for the modulation of gene expression (e.g., repressors,
stimulatory factors, etc.), signals involved in translation,
signals involved in modification of nucleic acids (e.g., by
methylation), etc. In the case of eukaryotic-type cells, further
regulatory control elements include signals involved in splicing,
post-translational modification, etc.
[0032] A further design procedure that may be applied is the design
of suitable cassettes to be combined to form a synthetic genome.
Upon generating synthetically a substantially exact copy of a
genome of known sequence, cassettes are selected which lie adjacent
to one another in that sequence and, preferably, which overlap one
another in order to facilitate the joining of the cassettes.
Factors to be considered in designing the cassettes include, e.g.,
that the segments be about 4 to 6.5 kb in length, not including
overlaps; that the segments contain only whole genes, except for
the overlaps; and that the overlaps with adjacent sequences are
about 200-250 (e.g., about 216) bp. Thus, each synthetic about 5 kb
piece is a cassette comprising one or more complete genes. An
illustration of cassettes that are designed, following these
constraints, for the synthesis of M. genitalium, is shown in FIG.
1.
[0033] In another embodiment, cassettes are designed to be
interchangeable, e.g., the cassettes are bounded by unique
sequences such as restriction enzyme or adaptor sites, which allow
the cassettes to be excised from the genome. The cassettes may be:
removed, manipulated (e.g., mutated) and returned to the original
location in the genome; substituted by other cassettes, such as
cassettes having functionally related genes; re-assorted
(rearranged) with other cassettes, for example in a combinatorial
fashion; etc. Mutations or other changes may be introduced, for
example, by inserting mutated nucleic acid from a natural source;
by site-directed mutagenesis, either in vivo or in vitro; by
synthesizing nucleic acids to contain a desired variation, etc.
[0034] As noted herein, genes of interest which directly or
indirectly lead to the production of desired products (e.g.,
therapeutic agents, biofuels, etc.) may be present in a synthetic
genome. To optimize the production of such products, the genes may
be manipulated and the effects of the manipulations evaluated by
introducing the modified synthetic genome into a biological system.
Features may be altered including, e.g., coding or regulatory
sequences, codon usage, adaptations for the use of a particular
growth medium, etc. Among the factors that may be evaluated are,
e.g., the amount of desired end product produced, tolerance to end
product, robustness, etc. Additional rounds of such manipulations
and assessments may be performed to further the optimization. Using
such iterative design and testing procedures (sometimes referred to
herein as "reiterative" or "recursive" improvement, "recursive
design," or "use of feedback loops") one may optimize the
production of a product of interest or may optimize growth of a
synthetic cell. One may make predictions about cellular behavior,
which may be confirmed or, if desired, modified. Furthermore, by
designing and manipulating genes in a synthetic genome according to
methods described herein, experimental studies may be performed,
e.g., to identify features that are important for the maintenance,
division, etc. of cells, features that are important to impart
"life" to an organism, etc.
[0035] A variety of methods may be used to generate and assemble
nucleic acid cassettes. As a first step, a cassette of interest is
generally subdivided into smaller portions from which it may be
assembled. Generally, the smaller portions are oligonucleotides of
about between about 30 nt and about 1 kb, e.g., about 50 nt (e.g.,
between about 45 and about 55). In one embodiment, the
oligonucleotides are designed so that they overlap adjacent
oligonucleotides, to facilitate their assembly into cassettes. For
example, for M. genitalium, the entire sequence may be divided into
a list of overlapping 48-mers with 24 nucleotide overlaps between
adjacent top and bottom oligonucleotides. An illustration of
suitable oligonucleotides for preparing M. genitalium is shown in
FIG. 1. The oligonucleotides may be synthesized using conventional
methods and apparatus, or they may be obtained from well-known
commercial suppliers.
[0036] Among the many methods which can be used to assemble
oligonucleotides to form longer molecules, such as the cassettes
described herein, are those described, e.g., in Stemmer et al.
(1995) (Gene 164, 49-53) and Young et al. (2004) (Nucleic Acids
Research 32, e59). One suitable method, called polymerase cycle
assembly (PCA), was used by Smith et al. (2003) (Proc Natl Acad Sci
USA 100, 15440-5) for the synthesis of the 5386 nt genome of
.PHI.X174. It is generally preferable to clone and/or amplify these
cassettes in order to generate enough material to manipulate
readily. In some embodiments, the cassettes are cloned and
amplified by conventional cell-based methods. In one embodiment,
e.g., when it is difficult to clone a cassette by conventional
cell-based methods, the cassettes are cloned in vitro. One such in
vitro method, which is discussed in co-pending U.S. Provisional
Patent Application Ser Nos. 60/675,850; 60/722,070; and 60/725,300,
uses rolling circle amplification, under conditions in which
background synthesis is significantly reduced.
[0037] Cassettes which may be generated according to various
exemplary methods may be of any suitable size. For example,
cassettes may range from about 1 kb to about 20 kb in length. A
convenient size is about 4 to about 7 kb, e.g., about 4.5 to about
6.5 kb, preferably about 5 kb. The term "about" with regard to a
particular polynucleotide length, as used herein, refers to a
polynucleotide that ranges from about 10% smaller than to about 10%
greater than the size of the polynucleotide. In order to facilitate
the assembly of cassettes, it is preferable that each cassette
overlaps the cassettes on either side, e.g., by at least about 50,
80, 100, 150, 200, 250 or 1300 nt. Larger constructs (up to the
size of, e.g., a minimal genome) comprising groups of such
cassettes are also included, and may be used in a modular fashion
according to various exemplary embodiments and methods.
[0038] A variety of methods may be used to assemble the cassettes.
For example, cassettes may be assembled in vitro, using methods of
recombination involving "chew-back" and repair steps, which employ
either 3' or 5' exonuclease activities, in a single step or in
multiple steps. Alternatively, the cassettes may be assembled with
an in vitro recombination system that includes enzymes from the
Dienocuccus radiodurans homologous recombination system. Methods of
in vivo assembly may also be used.
[0039] Example II describes the generation of a synthetic mouse
mitochondrial genome of 16.3 kb by the assembly of three cassettes.
Example II shows the design of 682 48-mer oligonucleotides, and the
assembly of those oligonucleotides into three overlapping segments
(cassettes). The oligonucleotides are then assembled into
cassettes, by such methods as the method described in Smith et al.
(2003), supra, modified in order to reduce heat damage to the
synthetic DNA.
[0040] According to one method, once a cassette is assembled, its
sequence may be verified. It is usually desirable to remove errors
which have arisen during the preparation of the cassettes, e.g.,
during the synthesis or assembly of the nucleic acid components.
Among the error correction methods which may be used are: (1)
methods to modify, tag and/or separate mismatched nucleotides so
that amplification errors may be prevented; (2) methods of global
error correction, using enzymes to recognize and cleave mismatches
in DNA, having known or unknown sequences, to produce fragments
from which the errors may be removed and the remaining error-free
pieces reassembled; (3) methods of site-directed mutagenesis; and
(4) methods to identify errors, select portions from independent
synthetic copies which are error-free, and assemble the error-free
portions, e.g., by overlap extension PCR (OE-PCR). Other methods to
recognize errors include, e.g., the use of isolated mismatch or
mutation recognition proteins, hybridization of
oligonucleotide-fluorescent probe conjugates, electrophoretic/DNA
chip methods, and differential chemical cleavage with reagents
assaying for base access ability either in solution or the solid
phase; such methods may be combined with conventional procedures to
remove errors.
[0041] In one embodiment, one or more identifying features, such as
a unique sequence (e.g., encoding a particular symbol or name, or,
e.g., spelling with the alphabet letter designations for the amino
acids) or an identifiable mutation which does not disrupt function
are introduced into the synthetic genome. Such sequences, sometimes
referred to herein as "watermarks," may serve not only to show that
the genome has, in fact, been artificially synthesized and to
enable branding and tracing, but also to distinguish the synthetic
genome from naturally occurring genomes. Often, genes or cassettes
contain selectable markers, such as drug resistance markers, which
aid in selecting nucleic acids that comprises the genes or
cassettes. The presence of such selectable markers may also
distinguish the synthetic genomes from naturally occurring nucleic
acids. A synthetic genome which is identical to a naturally
occurring genome, but which contains one or more identifying
markers as above, is sometimes referred to herein as being
"substantially identical" to the naturally occurring genome.
[0042] A synthetic genome according to one embodiment may be
present in any environment that allows for it to function. For
example, a synthetic genome may be present in (e.g., introduced
into) any of the biological systems described herein, or others.
The functions and activities of a synthetic genome, and the
consequences of modifying elements of the genome, can be studied in
a suitable biological system. Furthermore, a suitable biological
system allows proteins of interest (e.g., therapeutic agents) to be
produced. In some embodiments, if suitable substrates are provided,
downstream, non-proteinaceous products, such as energy sources
(e.g., hydrogen or ethanol) may also be produced, e.g., in
commercially useful amounts.
[0043] A variety of suitable biological systems may be used
according to various embodiments and methods. For example, in one
embodiment, a synthetic genome is contacted with a solution
comprising a conventional coupled transcription/translation system.
In such a system, the nucleic acid may be able to replicate itself,
or it may be necessary to replenish the nucleic acid, e.g.,
periodically.
[0044] In another embodiment, a synthetic genome is introduced into
a vesicle such that the genome is encapsulated by a protective
lipid-based material. In one embodiment, the synthetic genome is
introduced into a vesicle by contacting the synthetic genome,
optionally in the presence of desirable cytoplasmic elements such
complex organelles (e.g., ribosomes) and/or small molecules, with a
lipid composition or with a combination of lipids and other
components of functional cell membranes, under conditions in which
the lipid components encapsulate the synthetic genome and other
optional components to form a synthetic cell. In other embodiments,
a synthetic genome is contacted with a coupled
transcription/translation system and is then packaged into a
lipid-based vesicle. In a further embodiment, the internal
components are encapsulated spontaneously by the lipid
materials.
[0045] Exemplary embodiments also include a synthetic genome
introduced into a recipient cell, such as a bacterial cell, from
which some or all of the resident (original) genome has been
removed. For example, the entire resident genome may be removed to
form a ghost cell (a cell devoid of its functional natural genome)
and the resident genome may be replaced by the synthetic genome.
Alternatively, a synthetic genome may be introduced into a
recipient cell which contains some or all of its resident genome.
Following replication of the cell, the resident (original) and the
synthetic genome will segregate, and a progeny cell will form that
contains cytoplasmic and other epigenetic elements from the cell,
but that contains, as the sole genomic material, the synthetic
genome (e.g., a copy of a synthetic genome). Such a cell is a
synthetic cell according to various embodiments and methods, and
differs from the recipient cell in certain characteristics, e.g.,
nucleotide sequence, nucleotide source, or non-nucleotide
biochemical components.
[0046] A variety of in vitro methods may be used to introduce a
genome (synthetic, natural, or a combination thereof) into a cell.
These methods include, e.g., electroporation, lipofection, the use
of gene guns, etc. In one embodiment, a genome, such as a synthetic
genome, is immobilized in agar; and the agar plug is laid on a
liposome, which is then inserted into a host cell. In some
embodiments, a genome is treated to fold and compress before it is
introduced into a cell. Methods for inserting or introducing large
nucleic acid molecules, such as bacterial genomes, into a cell are
sometimes referred to herein as chromosome transfer, transport, or
transplantation.
[0047] According to one embodiment, a synthetic cell may comprise
elements from a host cell into which it has been introduced, e.g.,
a portion of the host genome, cytoplasm, ribosomes, membrane, etc.
In another embodiment, the components of a synthetic cell are
derived entirely from products encoded by the genes of the
synthetic genome and by products generated by those genes. Of
course, nutritive, metabolic and other substances as well as
physical conditions such as light and heat may be provided
externally to facilitate the growth, replication and expression of
a synthetic cell.
[0048] Various exemplary methods may be readily adapted to
computer-mediated and/or automated (e.g., robotic) formats. Many
synthetic genomes (including a variety of combinatorial variants of
a synthetic genome of interest) may be prepared and/or analyzed
simultaneously, using high throughput methods. Automated systems
for performing various methods as described herein are included. An
automated system permits design of a desired genome from genetic
components by selection using a bioinformatics computer system,
assembly and construction of numerous genomes and synthetic cells,
and automatic analysis of their characteristics, feeding back to
suggested design modifications.
[0049] While various embodiments and methods have been described
herein, it should be understood that they have been presented by
way of example only, and not limitation. Further, the breadth and
scope of a preferred embodiment should not be limited by any of the
above-described exemplary embodiments.
[0050] In the foregoing and in the following example, all
temperatures are set forth in uncorrected degrees Celsius; and,
unless otherwise indicated, all parts and percentages are by
weight.
EXAMPLE 1
Mycoplasma Comparative Genomics to Identify Genes in a Minimal Gene
Set
[0051] Mycoplasma Comparative Genomics. The 13 complete and 2
partial genome sequences currently in the inventors' dataset
comprise an in silico laboratory.
[0052] Comparisons of pairs of mycoplasma genomes using the whole
genome alignment tool MUMMER shows between species that are closely
related such as M. capricolum, M. mycoides SC, and Mesoplasma
florum, genome rearrangements are symmetrical about an axis passing
through the origins of replication and points that bisect the
genome equally. The direction of transcription of rearranged genes
almost always stays the same relative to the origin of replication.
This phenomenon has been observed for other species bacteria but
perhaps never so strikingly as with M. capricolum and M. mycoides
SC (see FIG. 2). These reciprocal crossovers suggest that any major
removal of DNA from one side of the genome might need to be matched
with a similar deletion from the other side so that the terminus of
replication remains constant.
[0053] Core Mycoplasma Gene Set. The 13 complete genome sequences
include 5 species from the pneumoniae branch of Mollicutes
phylogeny, 4 from the hominis branch, three from the
Entomoplasmatales branch and one from the Acholeplasmatales branch.
For each of the complete genomes we used BLASTp to generate
orthologs tables based on whether gene X in one genome has a
significant best BLASTp hit to gene Y in another genome, and Y is
X's best hit, then those genes are called orthologs. Using these
tables, we identified a core mycoplasma gene set, i.e., those
orthologous genes common to all 13 completely sequenced mycoplasma
species. Additionally, those tables identify orthologous gene sets
for three of the main mycoplasma tree branches.
[0054] The core mycoplasma gene set is .about.165 genes. That set
can be expanded to .about.200 by including those 45 genes missing
only in the intracellular parasite Phytoplasma asteris, which
obtains many of its essential metabolic products from the plant
cytoplasm this species lives in. The set can be further expanded to
.about.310 genes by taking into account non-orthologous gene
displacements that are obvious in some cases and suggested in
others. Obvious examples include the 14 genes absent in either or
both of the two non-glycolytic species Ureaplasma parvum or
Mycoplasma arthritides. An additional 96 genes are included in the
expanded core gene set because orthologs-are absent in only one of
the 12 complete genome (P. asteris is so different from the other
species it is usually ignored in this core set expansion process).
Based on this 13 genome comparative genomics analysis only, we
would predict that our model synthetic organism, M. laboratorium,
would need only about 310 genes and would have a genome containing
only about 372 kbp.
[0055] Given the significant evolutionary divergences of the 4
branches of mycoplasmas from each other because of their high rate
of evolution and different responses to gene loss, we also
determined the common gene sets for the pneumoniae, hominis and
Entomoplasmatales groups of mycoplasmas. (See Table 1, below). It
is instructive to consider an expanded core gene set for the 5
members of the pneumoniae group (which includes M. genitalium, our
planned platform for M. laboratorium construction). If one includes
only those genes present in at least 4 of the 5 group member
genomes; that expanded core set is 391 protein coding genes.
TABLE-US-00002 TABLE 1 Sizes of the Orthologous Gene Sets Shared by
All and Various Subgroups of the Mycoplasmas Orthologous gene sets
M. genitalium M. arthritldis M. capricolum Gene in all mycoplasmas
165 168 152 Genes in all mycoplasmas and B. subtilis and C.
perfringens 153 159 151 Genes in all mycoplasmas except Onion
Yellows Phytoplasma 200 206 197 Core mycoplasma genes lost in Onion
Yellows Phytoplasma 35 38 39 Genes present in all 5 pneumoniae
Glade members 294 -- -- Genes present in all 5 pneumoniae Glade
members and in hominis 220 220 -- group Genes present in all 5
pneumoniae clade members and in mycoides 244 -- 236 group Genes
present in all 5 pneumoniae Glade members and in P. asteris 207 --
-- Genes present in all 4 hominis Glade members 293 -- -- Genes
present in all hominis Glade members and in mycoides group 243 --
-- Genes present in all hominis clade members and in Phytoplasma
asteris -- 206 -- Genes present in all 3 mesoplasma/mycoides Glade
members -- -- 438 Genes present in all mycoides Glade members and
in Phytoplasma -- -- 230 asteris M. capricolum orthologs in M.
mycoides -- -- 715
EXAMPLE II
Synthesis of a Mouse Mitochondrial Genome
[0056] The mouse mitochondrial genome is a 16,299 bp circular DNA
and its sequence has been critically checked. We designed 682
48-mers to assemble it in three overlapping segments; Si (6,528
bp), S2 (5,328 bp), and S3 (4,508 bp) as diagrammed in FIG. 3A.
[0057] We assembled each of these three pieces by the method
described in Smith et al. (2003), supra, modified to reduce the
heating damage to the synthetic product. The gel shown in FIG. 3B
illustrates products from one such modified procedure that
dramatically reduces the time spent at high temperature.
[0058] The synthesis of three overlapping segments comprising the
entire mouse mitochondrial genome illustrates that combinations of
PCA and PCR can routinely assemble 5-6 Kris Prather segments of DNA
and validates our plan to build 5 Kris Prather cassettes for the
assembly of a cellular genome.
EXAMPLE III
Synthesis of 5 kb Cassettes of an Essential Region of the M.
Genitaliumgenome
[0059] 5 kb cassettes are constructed to generate a synthetic copy
of an essential region of the M. genitalium genome--the ribosomal
protein genes MG149.1 through MG181. This 18.5 kb region is flanked
by genes that tolerate transposon insertions (MG149 and MG182).
Sets of 386 top strand and 386 bottom strand oligonucleotides, of
48 nt, were synthesized to cover this region. These nucleic acids
are illustrated in FIG. 4.
[0060] The assembly of four overlapping segments (cassettes)
comprising these oligonucleotides is performed.
[0061] Using these techniques, cassettes of, for example, 4-6 kb
can be constructed that include gene sets of interest (e.g., a
minimal genome from a unicellular microorganism), and can be "mixed
and matched" with, or altered by substitutions from, e.g., other
species, to obtain a custom made genome, which can be introduced
into a vesicle or ghost cell for testing, as described above.
Synthetic cells thus constructed can be cultured under suitable
conditions to determine function. After determination of
functionality, the genome can be modified by substitution of
cassettes, and the process repeated until a desired result is
obtained.
[0062] From the foregoing description, one skilled in the art can
easily ascertain the essential characteristics of this invention,
and without departing from the spirit and scope thereof, can make
changes and modifications of the invention to adapt it to various
usage and conditions and to utilize the present invention to its
fullest extent. The preceding specific embodiments are to be
construed as merely illustrative, and not as limiting the scope of
the invention in any way whatsoever. The entire disclosure of all
applications, patents and publications cited above and in the
figures are hereby incorporated by reference.
Sequence CWU 1
1
3112DNAArtificial SequenceSynthetic construct 1ccaatgaaat gg
12212DNAArtificial SequenceSynthetic construct 2ccaagtctct gg
12312DNAArtificial SequenceSynthetic construct 3ccactgtgct gg
12
* * * * *