creation of a Bacterial cell controlled by a chemically synthesized Genome0

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Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

Daniel G.Gibson,1John I.Glass,1Carole Lartigue,1Vladimir N.Noskov,1Ray-Yuan Chuang,1Mikkel A.Algire,1Gwynedd A.Benders,2Michael G.Montague,1Li Ma,1Monzia M.Moodie,1Chuck Merryman,1Sanjay Vashee,1Radha Krishnakumar,1Nacyra Assad-Garcia,1Cynthia Andrews-Pfannkoch,1Evgeniya A.Denisova,1Lei Young,1Zhi-Qing Qi,1

Thomas H.Segall-Shapiro,1Christopher H.Calvey,1Prashanth P.Parmar,1Clyde A.Hutchison III,2Hamilton O.Smith,2J.Craig Venter 1,2*

We report the design,synthesis,and assembly of the 1.08–mega –base pair Mycoplasma mycoides JCVI-syn1.0genome starting from digitized genome sequence information and its transplantation into a M.capricolum recipient cell to create new M.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.I

n 1977,Sanger and colleagues determined the complete genetic sequence of phage ?X174(1),the first DNA genome to be completely sequenced.Eighteen years later,in 1995,our team was able to read the first complete genetic sequence of a self-replicating bacterium,Haemophilus influenzae (2).Reading the genetic sequence of a wide range of species has increased exponentially from these early studies.The ability to rapidly digitize genomic information has increased by more than eight orders of mag-nitude over the past 25years (3).Efforts to un-derstand all this new genomic information have spawned numerous new computational and experimental paradigms,yet our genomic knowl-edge 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 ge-netic repertoire?If so,can a complete genetic sys-tem be reproduced by chemical synthesis starting with only the digitized DNA sequence contained in a computer?

Our interest in synthesis of large DNA mol-ecules and chromosomes grew out of our efforts over the past 15years to build a minimal cell that contains only essential genes.This work was inaugurated in 1995when we sequenced the genome of Mycoplasma genitalium ,a bacterium with the smallest complement of genes of any known organism capable of independent growth in the laboratory.More than 100of the 485protein-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 synthe-sized DNA cassettes averaging about 6kb in size.This was accomplished through a combination of in vitro enzymatic methods and in vivo recombi-nation in Saccharomyces cerevisiae .The whole synthetic genome [582,970base pairs (bp)]was stably grown as a yeast centromeric plasmid (YCp)(7).

Several hurdles were overcome in transplanting and expressing a chemically synthesized chromo-some 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 ge-nome.Because 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 sub-species 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 com-mon 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 overcame this restriction barrier by methylat-ing 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-syn1.0genome,to create a new cell controlled by this synthetic genome.

Synthetic genome design.Design of the M.mycoides JCVI-syn1.0genome 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 trans-plantation of a genome that had been cloned and engineered in yeast,YCpMmyc1.1-D typeIIIres [GenBank accession CP001668](8).This project was critically dependent on the accuracy of these sequences.Although we believe that both fin-ished M.mycoides genome sequences are reli-able,there are 95sites 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 on the CP001621sequence (11).When it was finished,we chose 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 ap-peared biologically significant between CP001668and previously synthesized cassettes were cor-rected to match it exactly (11).Sequence differences between our synthetic cassettes and CP001668that occurred at 19sites appeared harmless and so were not corrected.These provide 19polymorphic differences between our synthetic genome (JCVI-syn1.0)and the natural (nonsynthetic)ge-nome (YCpMmyc1.1)that we have cloned in yeast and use as a standard for genome trans-plantation from yeast (8).To further differentiate between the synthetic genome and the natural one,we designed four watermark sequences (fig.S1)to replace one or more cassettes in regions experi-mentally demonstrated [watermarks 1(1246bp)and 2(1081bp)]or predicted [watermarks 3(1109bp)and 4(1222bp)]to not interfere with cell viability.These watermark sequences encode unique identifiers while limiting their translation into peptides.Table S1lists the differences be-tween the synthetic genome and this natural stan-dard.Figure S2shows a map of the M.mycoides JCVI-syn1.0genome.Cassette and assembly inter-mediate boundaries,watermarks,deletions,inser-tions,and genes of the M.mycoides JCVI syn1.0are shown in fig.S2,and the sequence of the transplanted mycoplasma clone sMmYCp235-1has been submitted to GenBank (accession CP002027).

Synthetic genome assembly strategy.The designed cassettes were generally 1080bp with 80-bp overlaps to adjacent cassettes (11).They were all produced by assembly of chemically

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1

The J.Craig Venter Institute,9704Medical Center Drive,Rockville,MD 20850,USA.2The J.Craig Venter Institute,10355Science Center Drive,San Diego,CA 92121,USA.

*To whom correspondence should be addressed.E-mail:jcventer@https://www.wendangku.net/doc/414706907.html,

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synthesized oligonucleotides by Blue Heron (Bothell,Washington).Each cassette was individ-ually synthesized and sequence-verified by the manufacturer.To aid in the building process,DNA cassettes and assembly intermediates were de-signed 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).A hierarchical strategy was designed to assemble the genome in three stages by transformation and homologous recombination in yeast from 10781-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 Escherichia

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 repre-sented (Fig.2A).In general,at least one 10-kb assembled fragment could be obtained by screening 10yeast clones.However,the rate of success varied from 10to 100%.All of the first-stage intermediates were sequenced.Nineteen out of 111assemblies 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 polymerase chain reaction (PCR)was performed on selected yeast clones (fig.S3and 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 ~105kb 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.Fortu-nately,these differences could easily be detected on an agarose gel before 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 11second-stage assemblies (11).As reported (14),circular plasmids the size of our second-stage assemblies could be isolated from yeast sphero-plasts after an alkaline-lysis procedure.To further purify the 11assembly intermediates,they were treated with exonuclease and passed through an anion-exchange column.A small fraction of the total plasmid DNA (1/100)was digested with Not I and analyzed by field-inversion gel electro-phoresis (FIGE)(Fig.2C).This method produced ~1m g of each assembly per 400ml of yeast culture (~1011cells).

The method above does not completely re-move all of the linear yeast chromosomal DNA,which we found could substantially decrease the yeast transformation and assembly efficiency.To further enrich for the 11circular assembly inter-mediates,~200ng 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 separated out of the agarose plug by electrophoresis,thus enriching for the trapped circular molecules.The 11circular 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 addi-tional vector sequence was not required because the yeast cloning elements were already present in assembly 811-900.

To screen for a complete genome,multiplex PCR was carried out with 11primer pairs,

A C G T

Elements for yeast propagation **Fig.1.The assembly of a synthetic M.mycoides genome in yeast.A synthetic M.mycoides genome was assembled from 1078overlapping DNA cassettes in three steps.In the first step,1080-bp cassettes (orange arrows),produced from overlapping synthetic oligonucleotides,were recombined in sets of 10to produce 109~10-kb assemblies (blue arrows).These were then recombined in sets of 10to produce 11~100-kb assemblies (green arrows).In the final stage of assembly,these 11fragments were recombined into the complete genome (red circle).With the exception of two 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 four watermarked regions (WM1to WM4),a 4-kb region that was intentionally deleted (94D),and elements for growth in yeast and genome transplantation.In addition,there are 20locations with nucleotide polymorphisms (asterisks).Coordinates of the genome are relative to the first nucleotide of the natural M.mycoides sequence.The designed sequence is 1,077,947bp.The locations of the Asc I and BssH II restriction sites are shown.Cassettes 1and 800-810were unnecessary and removed from the assembly strategy (11).Cassette 2overlaps cassette 1104,and cassette 799overlaps cassette 811.

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designed to span each of the 11100-kb assembly junctions (table S3).Of 48colonies screened,DNA extracted from one clone (sMmYCp235)produced all 11amplicons.PCR of the wild-type positive control (YCpMmyc1.1)produced an indistinguishable set of 11amplicons (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 pat-terns that are distinct from that of the natural M.mycoides genome (Figs.1and 3B).The sMmYCp235clone produced the restriction pattern expected for a completely assembled syn-thetic genome (Fig.3C).

Synthetic genome transplantation.Additional agarose plugs used in the gel analysis above (Fig.3C)were also used in genome transplantation ex-periments (11).Intact synthetic M.mycoides ge-nomes from the sMmYCp235yeast clone were transplanted into restriction-minus M.capricolum recipient cells,as described (8).Results were scored by selecting for growth of blue colonies on SP4medium containing tetracycline and X-gal at 37°C.Genomes isolated from this yeast clone produced 5to 15tetracycline-resistant blue colo-nies per agarose plug,a number comparable to that produced by the YCpMmyc1.1control.Recovery of colonies in all transplantation experiments was dependent on the presence of both M.capricolum recipient cells and an M.mycoides genome.Semisynthetic genome assembly and trans-plantation.To aid in testing the functionality of each 100-kb synthetic segment,semisynthetic ge-nomes 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 se-quence these intermediates.We cloned 11overlap-ping natural 100-kb assemblies in yeast by using a previously described method (16).In 11parallel reactions,yeast cells were cotransformed with frag-mented M.mycoides genomic DNA (YCpMmyc 1.1)that averaged ~100kb 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 as-semblies.The semisynthetic genomes that were constructed contained between 2and 10of the 11100-kb synthetic subassemblies (Table 1).The production of viable colonies produced after trans-plantation confirmed 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 811-820clone was used to produce a synthetic genome that did not transplant.This was expected because the error was a single –base pair deletion that creates a

frameshift in dnaA ,an essential gene for chromo-somal replication.We were previously unaware of this mutation.By using a semisynthetic genome construction strategy,we pinpointed 811-900as the source for failed synthetic transplantation experiments.Thus,we began to reassemble an error-free 811-900assembly,which was used to

produce the sMmYCp235yeast strain.The dnaA -mutated genome differs by only 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 811-900level by genome engineering in yeast (17).A repaired 811-900

Fig.2.Analysis of the assembly intermediates.

(A )Not I and Sbf I double restriction digestion anal-ysis of assembly 341-350

purified from E.coli .These restriction enzymes release the vector frag-ments (5.5and 3.4kb)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).(B )Analy-sis 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 4.5V/cm

for 3hours.S indicates the BAC-Tracker supercoiled DNA ladder (Epicentre).(C )Not I restriction digestion analysis of the 11~100-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.l indicates the lambda ladder (NEB).(D )Analysis of the 11pooled assemblies shown in (C)following topological trapping of the circular DNA and Not I digestion.One-fortieth of the DNA used to transform yeast is

represented.

10 kb

3 kb

4 kb 6 kb M 8 kb 10 kb assembly

(341-350)vector A 100 kb assembly (501-600)Linear yeast DNA

120 kb S B 95.7100.195.2100.1100.1100.1100.299.188.196.1104.1

2-100101-200W M 3201-300W M 1301-400401-500W M 2501-600601-700W M 4701-799811-900901-1000 94D 1001-1104λ48.5 kb

97 kb 146 kb

Expected Size (kb)C λD

1/40t h i n p u

t

pooled A L

W T

300 bp

500 bp 700 bp 900 bp 1200 bp 1

2345-611]7-10

235

B

1

23

4

5

6

78-910

W T -B s s H I I

W T -A s c I

235-B s s H I I

235-A s c I

H -A s c I

H -B s s H I I

λ

C

340 kb 437 kb 534 kb

631 kb 728 kb 48.5 kb

146 kb 243 kb 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 11amplicons;one at each of the 11assembly junctions.Yeast clone sMmYCp235(235)produced the 11PCR products expected for a complete genome assembly.For comparison,the natural genome extracted from yeast (WT,wild type)was also analyzed.PCR products were separated on a 2%E-gel (Invitrogen).L indicates the 100-bp ladder (NEB).(B )The sizes of the expected Asc I and BssH II restriction fragments for natural (WT)and synthetic (Syn235)M.mycoides genomes.(C )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 (B).

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assembly was used in a final-stage assembly to produce a yeast clone with a repaired genome.This yeast clone is named sMmYCP142and could be transplanted.A complete list of ge-nomes that have been assembled from 11pieces 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.3C),was performed.The restriction pattern ob-tained was consistent with a transplant produced from a synthetic M.mycoides genome (Fig.4B).

A single transplant originating from the sMmYCp235synthetic 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 poly-morphisms,eight new single-nucleotide poly-morphisms,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 IS1infected the 10-kb subassembly following its trans-fer to E.coli .The IS1insert 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 nonhomologous 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 be-longing to M.capricolum .This indicates that there

was a complete replacement of the M.capricolum genome by our synthetic genome during the trans-plant process.

The cells with only the synthetic genome are self-replicating and capable of logarithmic growth.Scanning and transmission electron micrographs (EMs)of M.mycoides JCVI-syn1.0cells show small,ovoid cells surrounded by cytoplasmic mem-branes (Fig.5,C to F).Proteomic analysis of M.mycoides JCVI-syn1.0and the wild-type control (YCpMmyc1.1)by two-dimensional gel electro-phoresis revealed almost identical patterns of protein spots (fig.S4)that differed from those pre-viously reported for M.capricolum (10).Fourteen genes are deleted or disrupted in the M.mycoides JCVI-syn1.0genome;however,the rate of appear-ance of colonies on agar plates and the colony morphology are similar (compare Fig.5,A and B).We did observe slight differences in the growth rates in a color-changing unit assay,with the JCVI-syn1.0transplants growing slightly faster than the MmcyYCp1.1control strain (fig.S6).

Discussion.In 1995,the quality standard for sequencing was considered to be one error in 10,000bp,and the sequencing of a microbial ge-nome required months.Today,the accuracy is sub-stantially higher.Genome coverage of 30to 50×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 more than 1million in an essential gene rendered the genome inactive,whereas major genome insertions and deletions in nonessential parts of the genome had no observable effect on viability.The demonstration that our synthetic genome gives rise to transplants with the characteristics of M.mycoides cells implies that the DNA sequence on 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 various other approaches to genome en-gineering that modify natural genomes by introduc-ing multiple insertions,substitutions,or deletions (18–22).This work provides a proof of principle for producing cells based on computer-designed genome sequences.DNA sequencing of a cel-lular genome allows storage of the genetic in-structions for life as a digital file.The synthetic genome described here has only limited modifi-cations from the naturally occurring M.mycoides genome.However,the approach we have de-veloped 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 cyto-plasm of the recipient cell is not synthetic.Pheno-typic effects of the recipient cytoplasm are diluted with protein turnover and as cells carrying only the

Table 1.Genomes that have been assembled from 11pieces 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,watermarked assembly.

Genome assembly

Synthetic fragments

Natural fragments Reconstituted natural genome None 1–11

2/11semisynthetic genome with one watermark 5WM,101–4,6–9,118/11semisynthetic genome without watermarks 1–4,6–8,115,9,109/11semisynthetic genome without watermarks 1–4,6–8,10–11

5,99/11semisynthetic genome with three watermarks 1,2WM,3WM,4,6,7WM,8,10–115,910/11semisynthetic genome with three watermarks 1,2WM,3WM,4,5WM,6,7WM,8,10–11911/11synthetic genome,

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

811-900correction of dnaA

1,2WM,3WM,4,5WM,6,7WM,8,9–11

None

Fig.4.Characterization of the transplants.(A )Trans-plants containing a synthetic genome were screened by mul-tiplex PCR with a primer set

that produces four amplicons,one internal to each of the four watermarks.One trans-plant (syn1.0)originating from

yeast clone sMmYCp235was analyzed alongside a natural,A L

W T s y n 1.0

100 bp 300 bp 500 bp 700 bp WM1WM2WM3

WM4B

340 kb 437 kb 534 kb

631 kb 728 kb

B s s H I I

A s c I

syn1.0WT A s c I

B s s H I I

1234

5678-91048.5 kb

λ146 kb 243 kb

nonsynthetic genome (WT)transplanted out of yeast.The

transplant containing the synthetic genome produced the four PCR products,whereas the WT genome did not produce

any.PCR products were separated on a 2%E-gel (Invitrogen).(B )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.

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transplanted genome replicate.Following transplan-tation and replication on a plate to form a colony (>30divisions 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 to produce synthetic cells renders 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 chromo-some in this and our previous study (7).

If the methods described here can be gener-alized,design,synthesis,assembly,and trans-plantation of synthetic chromosomes will no longer be a barrier to the progress of synthetic biology.We expect that the cost of DNA syn-thesis will follow what has happened with DNA sequencing and continue to exponentially de-crease.Lower synthesis costs combined with auto-mation 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 ap-plications expand,we anticipate that this work will continue to raise philosophical issues that have broad societal and ethical implications.We en-courage the continued discourse.

References and Notes

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

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

3.J.C.Venter,Nature 464,676(2010).

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

5.J.I.Glass et al .,Proc.Natl.Acad.Sci.U.S.A.103,425(2006).

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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,https://www.wendangku.net/doc/414706907.html,ler,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

https://www.wendangku.net/doc/414706907.html,/cgi/content/full/science.1190719/DC1Materials and Methods Figs.S1to S6Tables S1to S7References

9April 2010;accepted 13May 2010Published online 20May 2010;10.1126/science.1190719

Include this information when citing this paper.

Fig.5.Images of M.mycoides JCVI-syn1.0and WT M.mycoides .To compare the pheno-type of the JCVI-syn1.0and non-YCp WT strains,we examined colony mor-phology by plating cells

on SP4agar plates con-taining X-gal.Three days

after plating,the JCVI-syn1.0colonies are blue because the cells contain the lacZ gene and express b -galactosidase,which converts the X-gal to a blue compound (A ).The WT cells do not contain lacZ and remain white (B ).Both cell types have the fried egg colony mor-100 μm 100 μm 500 nm A B F

E D phology characteristic of most mycoplasmas.EMs were made of the JCVI-syn1.0isolate using two methods.(C )For scanning EM,samples were postfixed in osmium tetroxide,dehydrated and critical point dried with CO 2,and visualized with a Hitachi SU6600SEM at 2.0keV.(D )Negatively stained transmission EMs of dividing cells with 1%uranyl acetate on pure carbon substrate visualized using JEOL 1200EX CTEM at 80keV.To examine cell morphology,we compared uranyl acetate –stained EMs of M.mycoides

JCVI-syn1.0cells (E )with EMs of WT cells made in 2006that were stained with ammonium molybdate (F ).Both cell types show the same ovoid morphology and general appearance.EMs were provided by T.Deerinck and M.Ellisman of the National Center for Microscopy and Imaging Research at the University of California at San Diego.

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