[News] 1st cell with synthetic genome

[Michaelangelica]

Combining a series of techniques developed since 1995, J. Craig Venter and colleagues at the J. Craig Venter Institute in Rockville, Maryland began with a digitized genome sequence of Mycoplasma mycoides, a fast-growing bacterium with a 1-million-base genome. They ordered the pieces of that genome from a DNA sequence manufacturer, then used yeast to stitch the pieces together into a whole genome. The researchers transferred the synthetic M. mycoides genome into a M. capricolum recipient cell, replacing the native DNA, and the cell successfully booted up the new genome. The finished product was capable of replication and had all the expected properties of a M. mycoides cell.

 

"They are living cells," Venter told The Scientist. "The only difference is they have no natural history. Their parents were the computer."

 

The effort cost an estimated $40 million, with 20 people working for more than a decade, according to Science

 

Read more: 1st cell with synthetic genome - The Scientist - Magazine of the Life Sciences 1st cell with synthetic genome - The Scientist - Magazine of the Life Sciences

 

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We report the design, synthesis and assembly of the 1.08-

Mbp Mycoplasma mycoides JCVI-syn1.0 genome starting

from digitized genome sequence information and its

transplantation into a Mycoplasma capricolum recipient

cell to create new Mycoplasma mycoides cells that are

controlled only by the synthetic chromosome. The only

DNA in the cells is the designed synthetic DNA sequence,

including “watermark” sequences and other designed

gene deletions and polymorphisms, and mutations

acquired during the building process. The new cells have

expected phenotypic properties and are capable of

continuous self-replication.

In 1977, Sanger and colleagues determined the complete

genetic code of phage φX174 (1), the first DNA genome to be

completely sequenced. Eighteen years later, in 1995, our team

was able to read the first complete genetic code of a self-

replicating bacterium, Haemophilus influenzae (2). Reading

the genetic code of a wide range of species has increased

exponentially from these early studies. Our ability to rapidly

digitize genomic information has increased by more than

eight orders of magnitude over the past 25 years (3). Efforts

to understand all this new genomic information have spawned

numerous new computational and experimental paradigms,

yet our genomic knowledge remains very limited. No single

cellular system has all of its genes understood in terms of

their biological roles. Even in simple bacterial cells, do the

chromosomes contain the entire genetic repertoire? If so, can

a complete genetic system be reproduced by chemical

synthesis starting with only the digitized DNA sequence

contained in a computer?

Our interest in synthesis of large DNA molecules and

chromosomes grew out of our efforts over the past 15 years to

build a minimal cell that contains only essential genes. This

work was inaugurated in 1995 when we sequenced the

genome from Mycoplasma genitalium, a bacterium with the

smallest complement of genes of any known organism

capable of independent growth in the laboratory. More than

100 of the 485 protein-coding genes of M. genitalium are

dispensable when disrupted one-at-a-time (4–6).

We developed a strategy for assembling viral sized pieces

to produce large DNA molecules that enabled us to assemble

a synthetic M. genitalium genome in four stages from

chemically synthesized DNA cassettes averaging about 6 kb

in size. This was accomplished through a combination of in

vitro enzymatic methods and in vivo recombination in

Saccharomyces cerevisiae. The whole synthetic genome

(582,970 bp) was stably grown as a yeast centromeric

plasmid (YCp) (7).

Several hurdles were overcome in transplanting and

expressing a chemically synthesized chromosome in a

recipient cell. We needed to improve methods for extracting

intact chromosomes from yeast. We also needed to learn how

to transplant these genomes into a recipient bacterial cell to

establish a cell controlled only by a synthetic genome. Due to

the fact that M. genitalium has an extremely slow growth rate,

we turned to two faster growing mycoplasma species, M.

mycoides subspecies capri (GM12) as donor, and M.

capricolum subspecies capricolum (CK) as recipient.

To establish conditions and procedures for transplanting

the synthetic genome out of yeast, we developed methods for

cloning entire bacterial chromosomes as centromeric

plasmids in yeast, including a native M. mycoides genome (8,

9). However, initial attempts to extract the M. mycoides

genome from yeast and transplant it into M. capricolum

failed. We discovered that the donor and recipient

mycoplasmas share a common restriction system. The donor

genome was methylated in the native M. mycoides cells and

was therefore protected against restriction during the

transplantation from a native donor cell (10). However, the

bacterial genomes grown in yeast are unmethylated and so are

not protected from the single restriction system of the

recipient cell. We were able to overcome this restriction

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Daniel G. Gibson,1 John I. Glass,1 Carole Lartigue,1 Vladimir N. Noskov,1 Ray-Yuan Chuang,1 Mikkel A.

Algire,1 Gwynedd A. Benders,2 Michael G. Montague,1 Li Ma,1 Monzia M. Moodie,1 Chuck Merryman,1

Sanjay Vashee,1 Radha Krishnakumar,1 Nacyra Assad-Garcia,1 Cynthia Andrews-Pfannkoch,1 Evgeniya A.

Denisova,1 Lei Young,1 Zhi-Qing Qi,1 Thomas H. Segall-Shapiro,1 Christopher H. Calvey,1 Prashanth P.

Parmar,1 Clyde A. Hutchison III,2 Hamilton O. Smith,2 J. Craig Venter1,2*

1

The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. 2The J. Craig Venter Institute, 10355

Science Center Drive, San Diego, CA 92121, USA.

*

To whom correspondence should be addressed. E-mail: [email protected]

 

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barrier by methylating the donor DNA with purified

methylases or crude M. mycoides or M. capricolum extracts,

or by simply disrupting the recipient cell’s restriction system

(8).

We now have combined all of our previously established

procedures and report the synthesis, assembly, cloning, and

successful transplantation of the 1.08-Mbp M. mycoides

JCVI-syn1.0 genome, to create a new cell controlled by this

synthetic genome.

 

Results

Synthetic genome design

Design of the M. mycoides JCVI-syn1.0 genome was based

on the highly accurate finished genome sequences of two

laboratory strains of M. mycoides subspecies capri GM12 (8,

9) (11). One was the genome donor used by Lartigue et al.

[GenBank accession CP001621] (10). The other was a strain

created by transplantation of a genome that had been cloned

and engineered in yeast, YCpMmyc1.1-ΔtypeIIIres,

[GenBank accession CP001668] (8). This project was

critically dependent on the accuracy of these sequences.

Although we believe that both finished M. mycoides genome

sequences are reliable, there are 95 sites at which they differ.

We began to design the synthetic genome before both

sequences were finished. Consequently, most of the cassettes

were designed and synthesized based upon the CP001621

sequence (11). When it was finished, we chose to use the

sequence of the genome successfully transplanted from yeast

(CP001668) as our design reference (except that we kept the

intact typeIIIres gene). All differences that appeared

biologically significant between CP001668 and previously

synthesized cassettes were corrected to match it exactly (11).

Sequence differences between our synthetic cassettes and

CP001668 that occurred at 19 sites appeared harmless, and so

were not corrected. These provide 19 polymorphic

differences between our synthetic genome (JCVI-syn1.0) and

the natural (non-synthetic) genome (YCpMmyc1.1) that we

have cloned in yeast and use as a standard for genome

transplantation from yeast (8). To further differentiate

between the synthetic genome and the natural one, four

watermark sequences (fig. S1) were designed to replace one

or more cassettes in regions experimentally demonstrated

(watermarks 1 [1246 bp] and 2 [1081 bp]) or predicted

(watermarks 3 [1109 bp] and 4 [1222 bp]) to not interfere

with cell viability. These watermark sequences encode unique

identifiers while limiting their translation into peptides. Table

S1 lists the differences between the synthetic genome and this

natural standard. Figure S2 shows a map of the M. mycoides

JCVI-syn1.0 genome. Cassette and assembly intermediate

boundaries, watermarks, deletions, insertions, and genes of

the M. mycoides JCVI syn1.0 are shown in fig. S2, and the

sequence of the transplanted mycoplasma clone

sMmYCp235-1 has been submitted to GenBank (accession #

CP002027).

 

pSynthetic genome assembly strategy

The designed cassettes were generally 1,080 bp with 80-bp

overlaps to adjacent cassettes (11). They were all produced by

assembly of chemically synthesized oligonucleotides by Blue

Heron; Bothell, Washington. Each cassette was individually

synthesized and sequence-verified by the manufacturer. To

aid in the building process, DNA cassettes and assembly

intermediates were designed to contain Not I restriction sites

at their termini, and recombined in the presence of vector

elements to allow for growth and selection in yeast (7) (11).

pA hierarchical strategy was designed to assemble the

genome in 3 stages by transformation and homologous

recombination in yeast from 1,078 one-kb cassettes (Fig. 1)

(12, 13).

Assembly of 10-kb synthetic intermediates. In the first

stage, cassettes and a vector were recombined in yeast and

transferred to E. coli (11). Plasmid DNA was then isolated

from individual E. coli clones and digested to screen for cells

containing a vector with an assembled 10-kb insert. One

successful 10-kb assembly is represented (Fig. 2a). In

general, at least one 10-kb assembled fragment could be

obtained by screening 10 yeast clones. However, the rate of

success varied from 10-100%. All of the first-stage

intermediates were sequenced. Nineteen out of 111

assemblies contained errors. Alternate clones were selected,

sequence-verified, and moved on to the next assembly stage

(11).

Assembly of 100-kb synthetic intermediates. The pooled

10-kb assemblies and their respective cloning vectors were

transformed into yeast as above to produce 100-kb assembly

intermediates (11). Our results indicated that these products

cannot be stably maintained in E. coli so recombined DNA

had to be extracted from yeast. Multiplex PCR was performed

on selected yeast clones (fig. S3 and table S2). Because every

10-kb assembly intermediate was represented by a primer pair

in this analysis, the presence of all amplicons would suggest

an assembled 100-kb intermediate. In general, 25% or more

of the clones screened contained all of the amplicons

expected for a complete assembly. One of these clones was

selected for further screening. Circular plasmid DNA was

extracted and sized on an agarose gel alongside a supercoiled

marker. Successful second-stage assemblies with the vector

sequence are approximately 105 kb in length (Fig. 2b). When

all amplicons were produced following multiplex PCR, a

second-stage assembly intermediate of the correct size was

usually produced. In some cases, however, small deletions

occurred. In other instances, multiple 10-kb fragments were

assembled, which produced a larger second-stage assembly

intermediate. Fortunately, these differences could easily be

 

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detected on an agarose gel prior to complete genome

assembly.

Complete genome assembly. In preparation for the final

stage of assembly, it was necessary to isolate microgram

quantities of each of the 11 second-stage assemblies (11). As

reported (14), circular plasmids the size of our second-stage

assemblies could be isolated from yeast spheroplasts after an

alkaline-lysis procedure. To further purify the 11 assembly

intermediates, they were exonuclease-treated and passed

through an anion-exchange column. A small fraction of the

total plasmid DNA (1/100th) was digested with Not I and

analyzed by field-inversion gel electrophoresis (FIGE) (Fig.

2c). This method produced ~1 μg of each assembly per 400

ml yeast culture (~1011 cells).

The method above does not completely remove all of the

linear yeast chromosomal DNA, which we found could

significantly decrease the yeast transformation and assembly

efficiency. To further enrich for the eleven circular assembly

intermediates, ~200 ng samples of each assembly were

pooled and mixed with molten agarose. As the agarose

solidifies, the fibers thread through and topologically “trap”

circular DNA (15). Untrapped linear DNA can then be

electrophoresed out of the agarose plug, thus enriching for the

trapped circular molecules. The eleven circular assembly

intermediates were digested with Not I so that the inserts

could be released. Subsequently, the fragments were

extracted from the agarose plug, analyzed by FIGE (Fig. 2d),

and transformed into yeast spheroplasts (11). In this third and

final stage of assembly, an additional vector sequence was not

required since the yeast cloning elements were already

present in assembly 811-900.

To screen for a complete genome, multiplex PCR was

carried out with 11 primer pairs, designed to span each of the

eleven 100-kb assembly junctions (table S3). Of 48 colonies

screened, DNA extracted from one clone (sMmYCp235)

produced all 11 amplicons. PCR of the wild type (WT)

positive control (YCpMmyc1.1) produced an

indistinguishable set of 11 amplicons (Fig. 3a). To further

demonstrate the complete assembly of a synthetic M.

mycoides genome, intact DNA was isolated from yeast in

agarose plugs and subjected to two restriction analyses; Asc I

and BssH II (11). Because these restriction sites are present in

three of the four watermark sequences, this choice of

digestion produces restriction patterns that are distinct from

the natural M. mycoides genome (Figs. 1 and 3b). The

sMmYCp235 clone produced the restriction pattern expected

for a completely assembled synthetic genome (Fig. 3c).

 

pSynthetic genome transplantation

Additional agarose plugs used in the gel analysis above (Fig.

3c) were also used in genome transplantation experiments

(11). Intact synthetic M. mycoides genomes from the

sMmYCp235 yeast clone were transplanted into restriction-

minus M. capricolum recipient cells, as described (8). Results

were scored by selecting for growth of blue colonies on SP4

medium containing tetracycline and X-gal at 37 °C. Genomes

isolated from this yeast clone produced 5-15 tetracycline-

resistant blue colonies per agarose plug. This was comparable

to the YCpMmyc1.1 control. Recovery of colonies in all

transplantation experiments was dependent on the presence of

both M. capricolum recipient cells and an M. mycoides

genome.

 

Semi-synthetic genome assembly and transplantation

To aid in testing the functionality of each 100-kb synthetic

segment, semi-synthetic genomes were constructed and

transplanted. By mixing natural pieces with synthetic ones,

the successful construction of each synthetic 100-kb assembly

could be verified without having to sequence these

intermediates. We cloned 11 overlapping natural 100-kb

assemblies in yeast by using a previously described method

(16). In 11 parallel reactions, yeast cells were co-transformed

with fragmented M. mycoides genomic DNA (YCpMmyc

1.1) that averaged ~100 kb in length and a PCR-amplified

vector designed to overlap the ends of the 100-kb inserts. To

maintain the appropriate overlaps so that natural and synthetic

fragments could be recombined, the PCR-amplified vectors

were produced via primers with the same 40-bp overlaps used

to clone the 100-kb synthetic assemblies. The semi-synthetic

genomes that were constructed contained between two and

ten of the eleven 100-kb synthetic subassemblies (Table 1).

The production of viable colonies produced after

transplantation, ionfirmed that the synthetic fraction of each

genome contained no lethal mutations. Only one of the 100-

kb subassemblies, 811-900, was not viable.

Initially, an error-containing 811-820 clone was used to

produce a synthetic genome that did not transplant. This was

expected since the error was a single base pair deletion that

creates a frameshift in dnaA, an essential gene for

chromosomal replication. We were previously unaware of

this mutation. By using a semi-synthetic genome construction

strategy, we were able to pinpoint 811-900 as the source for

failed synthetic transplantation experiments. Thus, we began

to reassemble an error-free 811-900 assembly, which was

used to produce the sMmYCp235 yeast strain. The dnaA-

mutated genome only differs by one nucleotide from the

synthetic genome in sMmYCp235. This genome served as a

negative control in our transplantation experiments. The dnaA

mutation was also repaired at the 811-900 level by genome

engineering in yeast (17) . A repaired 811-900 assembly was

used in a final stage assembly to produce a yeast clone with a

repaired genome. This yeast clone is named sMmYCP142

and could be transplanted. A complete list of genomes that

 

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have been assembled from 11 pieces and successfully

transplanted is provided in Table 1.

 

Characterization of the synthetic transplants

To rapidly distinguish the synthetic transplants from M.

capricolum or natural M. mycoides, two analyses were

performed. First, four primer pairs that are specific to each of

the four watermarks were designed such that they produce

four amplicons in a single multiplex PCR reaction (table S4).

All four amplicons were produced by transplants generated

from sMmYCp235, but not YCpMmyc1.1 (Fig. 4a). Second,

the gel analysis with Asc I and BssH II, described above (Fig.

3d), was performed. The restriction pattern obtained was

consistent with a transplant produced from a synthetic M.

mycoides genome (Fig. 4b).

A single transplant originating from the sMmYCp235

synthetic genome was sequenced. We refer to this strain as M.

mycoides JCVI-syn1.0. The sequence matched the intended

design with the exception of the known polymorphisms, 8

new single nucleotide polymorphisms, an E. coli transposon

insertion, and an 85-bp duplication (table S1). The transposon

insertion exactly matches the size and sequence of IS1, a

transposon in E. coli. It is likely that IS1 infected the 10-kb

sub-assembly following its transfer to E. coli. The IS1 insert

is flanked by direct repeats of M. mycoides sequence

suggesting that it was inserted by a transposition mechanism.

The 85-bp duplication is a result of a non-homologous end

joining event, which was not detected in our sequence

analysis at the 10-kb stage. These two insertions disrupt two

genes that are evidently non-essential. We did not find any

sequences in the synthetic genome that could be identified as

belonging to M. capricolum. This indicates that there was a

complete replacement of the M. capricolum genome by our

synthetic genome during the transplant process.

The cells with only the synthetic genome are self

replicating and capable of logarithmic growth. Scanning and

transmission electron micrographs (EM) of M. mycoides

JCVI-syn1.0 cells show small, ovoid cells surrounded by

cytoplasmic membranes (Fig. 5c-5f). Proteomic analysis of

M. mycoides JCVI-syn1.0 and the WT control

(YCpMmyc1.1) by two-dimensional gel electrophoresis

revealed almost identical patterns of protein spots (fig. S4)

and these were clearly different from those previously

reported for M. capricolum (10). Fourteen genes are deleted

or disrupted in the M. mycoides JCVI-syn1.0 genome,

however the rate of appearance of colonies on agar plates and

the colony morphology are similar (compare Fig. 5a and .

We did observe slight differences in the growth rates in a

color changing unit assay, with the JCVI-syn1.0 transplants

growing slightly faster than the MmcyYCp1.1 control strain

(fig. S6).

 

Discussion

In 1995, the quality standard for sequencing was considered

to be one error in 10,000 bp and the sequencing of a

microbial genome required months. Today, the accuracy is

substantially higher. Genome coverage of 30-50X is not

unusual, and sequencing only requires a few days. However,

obtaining an error-free genome that could be transplanted into

a recipient cell to create a new cell controlled only by the

synthetic genome was complicated and required many quality

control steps. Our success was thwarted for many weeks by a

single base pair deletion in the essential gene dnaA. One

wrong base out of over one million in an essential gene

rendered the genome inactive, while major genome insertions

and deletions in non-essential parts of the genome had no

observable impact on viability. The demonstration that our

synthetic genome gives rise to transplants with the

characteristics of M. mycoides cells implies that the DNA

sequence upon which it is based is accurate enough to specify

a living cell with the appropriate properties.

Our synthetic genomic approach stands in sharp contrast to

a variety of other approaches to genome engineering that

modify natural genomes by introducing multiple insertions,

substitutions, or deletions (18–22). This work provides a

proof of principle for producing cells based upon genome

sequences designed in the computer. DNA sequencing of a

cellular genome allows storage of the genetic instructions for

life as a digital file. The synthetic genome described in this

paper has only limited modifications from the naturally

occurring M. mycoides genome. However, the approach we

have developed should be applicable to the synthesis and

transplantation of more novel genomes as genome design

progresses (23).

We refer to such a cell controlled by a genome assembled

from chemically synthesized pieces of DNA as a “synthetic

cell”, even though the cytoplasm of the recipient cell is not

synthetic. Phenotypic effects of the recipient cytoplasm are

diluted with protein turnover and as cells carrying only the

transplanted genome replicate. Following transplantation and

replication on a plate to form a colony (>30 divisions or >109

fold dilution), progeny will not contain any protein molecules

that were present in the original recipient cell (10, 24). This

was previously demonstrated when we first described genome

transplantation (10). The properties of the cells controlled by

the assembled genome are expected to be the same as if the

whole cell had been produced synthetically (the DNA

software builds its own hardware).

The ability tp produce synthetic cells renders it it essential

for researchers making synthetic DNA constructs and cells to

clearly watermark their work to distinguish it from naturally

occurring DNA and cells. We have watermarked the synthetic

chromosome in this and our previous study (7).

 

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If the methods described here can be generalized, design,

synthesis, assembly, and transplantation of synthetic

chromosomes will no longer be a barrier to the progress of

synthetic biology. We expect that the cost of DNA synthesis

will follow what has happened with DNA sequencing and

continue to exponentially decrease. Lower synthesis costs

combined with automation will enable broad applications for

synthetic genomics.

We have been driving the ethical discussion concerning

synthetic life from the earliest stages of this work (25, 26). As

synthetic genomic applications expand, we anticipate that this

work will continue to raise philosophical issues that have

broad societal and ethical implications. We encourage the

continued discourse.

References and Notes

1. F. Sanger et al., Nature 265, 687 (Feb 24, 1977).

2. R. D. Fleischmann et al., Science 269, 496 (Jul 28, 1995).

3. J. C. Venter, Nature 464, 676 (Apr 1).

4. C. A. Hutchison et al., Science 286, 2165 (Dec 10, 1999).

5. J. I. Glass et al., Proc Natl Acad Sci U S A 103, 425 (Jan

10, 2006).

6. H. O. Smith, J. I. Glass, C. A. Hutchison III, J. C. Venter,

in Accessing Uncultivated Microorganisms: From the

Environment to Organisms and Genomes and Back K.

Zengler, Ed. (ASM Press, Washington, 2008), pp. 320.

7. D. G. Gibson et al., Science 319, 1215 (Feb 29, 2008).

8. C. Lartigue et al., Science 325, 1693 (Sep 25, 2009).

9. G. A. Benders et al., Nucleic Acids Res, (Mar 7, 2010).

10. C. Lartigue et al., Science 317, 632 (Aug 3, 2007).

11. Supplementary information is available on Science

Online.

12. D. G. Gibson, Nucleic Acids Res 37, 6984 (Nov, 2009).

13. D. G. Gibson et al., Proc Natl Acad Sci U S A 105, 20404

(Dec 23, 2008).

14. R. J. Devenish, C. S. Newlon, Gene 18, 277 (Jun, 1982).

15. W. W. Dean, B. M. Dancis, C. A. Thomas, Jr., Anal

Biochem 56, 417 (Dec, 1973).

16. S. H. Leem et al., Nucleic Acids Res 31, e29 (Mar 15,

2003).

17. V. N. Noskov, T. H. Segall-Shapiro, R. Y. Chuang,

Nucleic Acids Res 38, 2570 (May 1).

18. M. Itaya, K. Tsuge, M. Koizumi, K. Fujita, Proc Natl

Acad Sci U S A 102, 15971 (Nov 1, 2005).

19. M. Itaya, FEBS Lett 362, 257 (Apr 10, 1995).

20. H. Mizoguchi, H. Mori, T. Fujio, Biotechnol Appl

Biochem 46, 157 (Mar, 2007).

21. J. Y. Chun et al., Nucleic Acids Res 35, e40 (2007).

22. H. H. Wang et al., Nature 460, 894 (Aug 13, 2009).

23. A. S. Khalil, J. J. Collins, Nat Rev Genet 11, 367 (May).

24. A mycoplasma cell, with a cell mass of about 10-13 g,

contains fewer than 106 molecules of protein. (If it

contains 20% protein this is 2 x 10-14 g protein per cell. At

a molecular weight of 120 Daltons per amino acid residue

each cell contains (2 x 10-14)/120 = 1.7 x 10-16 moles of

peptide residues. This is 1.7 x 10-16 x 6 x 1023 = 1 x 108

residues per cell. If the average size of a protein is 300

residues then a cell contains about 3 x 105 protein

molecules.) After 20 cell divisions the number of progeny

exceeds the total number of protein molecules present in

the recipient cell. So, following transplantation and

replication to form a colony on a plate, most cells will

contain no protein molecules that were present in the

original recipient cell.

25. M. K. Cho, D. Magnus, A. L. Caplan, D. McGee, Science

286, 2087 (Dec 10, 1999).

26. M. S. Garfinkel, D. Endy, G. E. Epstein, R. M. Friedman.

(2007).

27. D. G. Gibson et al., Nat Methods 6, 343 (May, 2009).

28. We thank Synthetic Genomics, Inc. for generous funding

of this work. We thank J. B. Hostetler, D. Radune, N. B.

Fedorova, M. D. Kim, B. J. Szczypinski, I. K. Singh, J. R.

Miller, S. Kaushal, R. M. Friedman, and J. Mulligan for

their contributions to this work. Electron micrographs

were generously provided by T. Deerinck and M. Ellisman

of the National Center for Microscopy and Imaging

Research at the University of California at San Diego.

J.C.V. is Chief Executive Officer and Co-Chief Scientific

Officer of SGI. H.O.S. is Co-Chief Scientific Officer and

on the Board of Directors of SGI. C.A.H. is Chairman of

the SGI Scientific Advisory Board. All three of these

authors and JCVI hold SGI stock. JCVI has filed patent

applications on some of the techniques described in this

paper.

 

Supporting Online Material

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome -- Gibson et al. 329 (5987): 52 Data Supplement - Supporting Online Material -- Science

Materials and Methods

Figs. S1 to S6

Tables S1 to S7

References

9 April 2010; accepted 13 May 2010

Published online 20 May 2010; 10.1126/science.1190719

Include this information when citing this paper.

Fig. 1. The assembly of a synthetic M. mycoides genome in

yeast. A synthetic M. mycoides genome was assembled from

1,078 overlapping DNA cassettes in three steps. In the first

step, 1,080-bp cassettes (orange arrows), produced from

overlapping synthetic oligonucleotides, were recombined in

sets of 10 to produce one hundred nine ~10-kb assemblies

(blue arrows). These were then recombined in sets of 10 to

produce eleven ~100-kb assemblies (green arrows). In the

final stage of assembly, these eleven fragments were

recombined into the complete genome (red circle). With the

 

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exception of 2 constructs that were enzymatically pieced

together in vitro (27) (white arrows), assemblies were carried

out by in vivo homologous recombination in yeast. Major

variations from the natural genome are shown as yellow

circles. These include 4 watermarked regions (WM1-WM4),

a 4-kb region that was intentionally deleted (94D), and

elements for growth in yeast and genome transplantation. In

addition, there are 20 locations with nucleotide

polymorphisms (asterisks). Coordinates of the genome are

relative to the first nucleotide of the natural M. mycoides

sequence. The designed sequence is 1,077,947 bp. The

locations of the Asc I and BssH II restriction sites are shown.

Cassettes 1 and 800-810 were unnecessary and removed from

the assembly strategy (11). Cassette 2 overlaps cassette 1104

and cassette 799 overlaps cassette 811.

Fig. 2. Analysis of the assembly intermediates. (a) Not I and

Sbf I double restriction digestion analysis of assembly 341-

350 purified from E. coli. These restriction enzymes release

the vector fragments (5.5 kb and 3.4 kb) from the 10-kb

insert. Insert DNA was separated from the vector DNA on a

0.8% E-gel (Invitrogen). M indicates the 1-kb DNA ladder

(New England Biolabs; NEB). (:hihi: Analysis of assembly 501-

600 purified from yeast. The 105-kb circles (100-kb insert

plus 5-kb vector) were separated from the linear yeast

chromosomal DNA on a 1% agarose gel by applying 4.5

V/cm for 3 hours. S indicates the BAC-Tracker supercoiled

DNA ladder (Epicentre). © Not I restriction digestion

analysis of the eleven ~100-kb assemblies purified from

yeast. These DNA fragments were analyzed by FIGE on a 1%

agarose gel. The expected insert size for each assembly is

indicated. λ indicates the lambda ladder (NEB). (d) Analysis

of the 11 pooled assemblies shown in © following

topological trapping of the circular DNA and Not I digestion.

One fortieth of the DNA used to transform yeast is

represented.

Fig. 3. Characterization of the synthetic genome isolated from

yeast. (a) Yeast clones containing a completely assembled

synthetic genome were screened by multiplex PCR with a

primer set that produces 11 amplicons; one at each of the 11

assembly junctions. Yeast clone sMmYCp235 (235)

produced the 11 PCR products expected for a complete

genome assembly. For comparison, the natural genome

extracted from yeast (WT) was also analyzed. PCR products

were separated on a 2% E-gel (Invitrogen). L indicates the

100-bp ladder (NEB). (:) The sizes of the expected Asc I and

BssH II restriction fragments for natural (WT) and synthetic

(Syn235) M. mycoides genomes. © Natural (WT) and

synthetic (235) M. mycoides genomes were isolated from

yeast in agarose plugs. In addition, DNA was purified from

the host strain alone (H). Agarose plugs were digested with

Asc I or BssH II and fragments were separated by clamped

homogeneous electrical field (CHEF) gel electrophoresis.

Restriction fragments corresponding to the correct sizes are

indicated by the fragment numbers shown in (;).

Fig. 4. Characterization of the transplants. (a) Transplants

containing a synthetic genome were screened by multiplex

PCR with a primer set that produces 4 amplicons; one internal

to each of the four watermarks. One transplant (syn1.0)

originating from yeast clone sMmYCp235 was analyzed

alongside a natural, non-synthetic genome (WT) transplanted

out of yeast. The transplant containing the synthetic genome

produced the 4 PCR products whereas the WT genome did

not produce any. PCR products were separated on a 2% E-gel

(Invitrogen). (;) Natural (WT) and synthetic (syn1.0) M.

mycoides genomes were isolated from M. mycoides

transplants in agarose plugs. Agarose plugs were digested

with Asc I or BssH II and fragments were separated by CHEF

gel electrophoresis. Restriction fragments corresponding to

the correct sizes are indicated by the fragment numbers

shown in Fig. 3b.

Fig. 5. Images of M. mycoides JCVI-syn1.0 and WT M.

mycoides. To compare the phenotype of the JCVI-syn1.0 and

non-YCp WT strains, we examined colony morphology by

plating cells on SP4 agar plates containing X-gal. Three days

after plating, the JCVI-syn1.0 colonies are blue because the

cells contain the lacZ gene and express beta-galactosidase,

which converts the X-gal to a blue compound (a). The WT

cells do not contain lacZ and remain white (:D. Both cell

types have the fried egg colony morphology characteristic of

most mycoplasmas. EMs were made of the JCVI-syn1.0

isolate using two methods. © For scanning EM, samples

were post-fixed in osmium tetroxide, dehydrated and critical

point dried with CO2, and visualized using a Hitachi SU6600

SEM at 2.0 keV. (d) Negatively stained transmission EMs of

dividing cells using 1% uranyl acetate on pure carbon

substrate visualized using JEOL 1200EX CTEM at 80 keV.

To examine cell morphology, we compared uranyl acetate

stained EMs of M. mycoides JCVI-syn1.0 cells (e) with EMs

of WT cells made in 2006 that were stained with ammonium

molybdate (f). Both cell types show the same ovoid

morphology and general appearance. EMs were provided by

Tom Deerinck and Mark Ellisman of the National Center for

Microscopy and Imaging Research at the University of

California at San Diego.

 

/ Science/AAAS | Science Magazine: Science Express / 20 May 2010 / Page 7 / 10.1126/science.1190719

 

Table 1. Genomes that have been assembled from 11 pieces and successfully transplanted. Assembly 2-100 = 1, assembly 101-

200 = 2, assembly 201-300 = 3, assembly 301-400 = 4, assembly 401-500 = 5, assembly 501-600 = 6, assembly 601-700 = 7,

assembly 701-799 = 8, assembly 811-900 = 9, assembly 901-1000 = 10, assembly 1001-1104 = 11. WM indicates watermarked

assembly.

 

Genome Assembly Synthetic Fragments Natural Fragments

Reconstituted natural genome None 1-11

2/11 semi-synthetic genome with 1 watermark 5WM, 10 1-4, 6-9, 11

8/11 semi-synthetic genome without watermarks 1-4, 6-8, 11 5, 9, 10

9/11 semi-synthetic genome without watermarks 1-4, 6-8, 10-11 5, 9

9/11 semi-synthetic genome with 3 watermarks 1, 2WM, 3WM, 4, 6, 7WM, 8, 10-11 5, 9

10/11 semi-synthetic genome with 3 watermarks 1, 2WM, 3WM, 4, 5WM, 6, 7WM, 8, 10-11 9

11/11 synthetic genome, 811-820 correction of dnaA 1, 2WM, 3WM, 4, 5WM, 6, 7WM, 8, 9-11 None

11/11 synthetic genome, 811-900 correction of dnaA 1, 2WM, 3WM, 4, 5WM, 6, 7WM, 8, 9-11 None

[HydrogenBond]

What this demonstrates, which I have been saying all along, is the cytoplasm has its own autonomy, since it can accommodate the new DNA. The DNA is like the hard drive of the cell. They simply changed the hard drive. I would guess, if they can get the cell to replicate, the DNA will gradually mutated toward a better equilibrium. This will occur since the new hard drive sets a potential with the cell and the cell will attempt to lower this.

 

In nature, interaction with the environment can alter the cell through material exchange which adds or takes away potential. This is similar in that the DNA now sets a potential with the new grid. There is a push to form equilibrium.

[Michaelangelica]

I would guess, if they can get the cell to replicate, the DNA will grad

Have they managed replication of the new cell?

I find this new biological research astounding