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Creation of a Bacterial Cell Controlled by a Chemically Synthesized
Genome
Daniel G. Gibson
Science 329
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5 article(s) on the ISI Web of Science This article has been 13 articles hosted by HighWire Press; see: This article has been This article appears in the following Genetics (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. 2010 by the American Association for the Advancement of Science; all rights reserved. The title registered trademark of AAAS. crude M. mycoides or M. capricolum extracts, orby simply disrupting the recipient cell's restriction Creation of a Bacterial Cell Controlled system(8).
We now have combined all of our previously established procedures and report the synthesis, by a Chemically Synthesized Genome assembly, cloning, and successful transplantation of the 1.08-Mbp M. mycoides JCVI-syn1.0genome, to create a new cell controlled by this Daniel G. Gibson,1 John I. Glass,1 Carole Lartigue,1 Vladimir N. Noskov,1 Ray-Yuan Chuang,1 synthetic genome.
Mikkel A. Algire,1 Gwynedd A. Benders,2 Michael G. Montague,1 Li Ma,1 Monzia M. Moodie,1 Synthetic genome design. Design of the M.
Chuck Merryman,1 Sanjay Vashee,1 Radha Krishnakumar,1 Nacyra Assad-Garcia,1 mycoides JCVI-syn1.0 genome was based on the Cynthia Andrews-Pfannkoch,1 Evgeniya A. Denisova,1 Lei Young,1 Zhi-Qing Qi,1 highly accurate finished genome sequences of two Thomas H. Segall-Shapiro,1 Christopher H. Calvey,1 Prashanth P. Parmar,1 Clyde A. Hutchison III,2 laboratory strains of M. mycoides subspecies capri Hamilton O. Smith,2 J. Craig Venter1,2* GM12 (8, 9, 11). One was the genome donor usedby Lartigue et al. [GenBank accession CP001621] We report the design, synthesis, and assembly of the 1.08–mega–base pair Mycoplasma mycoides (10). The other was a strain created by trans- JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation plantation of a genome that had been cloned and into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the engineered in yeast, YCpMmyc1.1-DtypeIIIres synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, [GenBank accession CP001668] (8). This project including "watermark" sequences and other designed gene deletions and polymorphisms, and was critically dependent on the accuracy of these mutations acquired during the building process. The new cells have expected phenotypic properties sequences. Although we believe that both fin- and are capable of continuous self-replication.
ished M. mycoides genome sequences are reli-able, there are 95 sites at which they differ. We In 1977, Sanger and colleagues determined Wedevelopedastrategyforassemblingviral- begantodesignthesyntheticgenomebeforeboth the complete genetic sequence of phage sized pieces to produce large DNA molecules that sequences were finished. Consequently, most of ϕX174 (1), the first DNA genome to be enabled us to assemble a synthetic M. genitalium the cassettes were designed and synthesized based on February 15, 2011 completely sequenced. Eighteen years later, in genome in four stages from chemically synthe- on the CP001621 sequence (11). When it was 1995, our team was able to read the first complete sized DNA cassettes averaging about 6 kb in size.
finished, we chose the sequence of the genome genetic sequence of a self-replicating bacterium, This was accomplished through a combination of successfully transplanted from yeast (CP001668) Haemophilus influenzae (2). Reading the genetic in vitro enzymatic methods and in vivo recombi- as our design reference (except that we kept the sequence of a wide range of species has increased nation in Saccharomyces cerevisiae. The whole intact typeIIIres gene). All differences that ap- exponentially from these early studies. The synthetic genome [582,970 base pairs (bp)] was peared biologically significant between CP001668 ability to rapidly digitize genomic information stably grown as a yeast centromeric plasmid and previously synthesized cassettes were cor- has increased by more than eight orders of mag- rected to match it exactly (11). Sequence differences nitude over the past 25 years (3). Efforts to un- Several hurdles were overcome in transplanting between our synthetic cassettes and CP001668 derstand all this new genomic information have and expressing a chemically synthesized chromo- that occurred at 19 sites appeared harmless and so spawned numerous new computational and some in a recipient cell. We needed to improve were not corrected. These provide 19 polymorphic experimental paradigms, yet our genomic knowl- methods for extracting intact chromosomes from differences between our synthetic genome edge remains very limited. No single cellular yeast. We also needed to learn how to transplant (JCVI-syn1.0) and the natural (nonsynthetic) ge- system has all of its genes understood in terms of these genomes into a recipient bacterial cell to nome (YCpMmyc1.1) that we have cloned in their biological roles. Even in simple bacterial establish a cell controlled only by a synthetic ge- yeast and use as a standard for genome trans- cells, do the chromosomes contain the entire ge- nome. Because M. genitalium has an extremely plantation from yeast (8). To further differentiate netic repertoire? If so, can a complete genetic sys- slow growth rate, we turned to two faster-growing between the synthetic genome and the natural one, tem be reproduced by chemical synthesis starting mycoplasma species, M. mycoides subspecies we designed four watermark sequences (fig. S1) to with only the digitized DNA sequence contained capri (GM12) as donor, and M. capricolum sub- replace one or more cassettes in regions experi- species capricolum (CK) as recipient.
mentally demonstrated [watermarks 1 (1246 bp) Our interest in synthesis of large DNA mol- To establish conditions and procedures for and 2 (1081 bp)] or predicted [watermarks 3 ecules and chromosomes grew out of our efforts transplanting the synthetic genome out of yeast, (1109 bp) and 4 (1222 bp)] to not interfere with over the past 15 years to build a minimal cell that we developed methods for cloning entire bacterial cell viability. These watermark sequences encode contains only essential genes. This work was chromosomes as centromeric plasmids in yeast, unique identifiers while limiting their translation inaugurated in 1995 when we sequenced the including a native M. mycoides genome (8, 9).
into peptides. Table S1 lists the differences be- genome of Mycoplasma genitalium, a bacterium However, initial attempts to extract the M.
tween the synthetic genome and this natural stan- with the smallest complement of genes of any mycoides genome from yeast and transplant it dard. Figure S2 shows a map of the M. mycoides known organism capable of independent growth into M. capricolum failed. We discovered that the JCVI-syn1.0 genome. Cassette and assembly inter- in the laboratory. More than 100 of the 485 donor and recipient mycoplasmas share a com- mediate boundaries, watermarks, deletions, inser- protein-coding genes of M. genitalium are mon restriction system. The donor genome was tions, and genes of the M. mycoides JCVI syn1.0 dispensable when disrupted one at a time (4–6).
methylated in the native M. mycoides cells and are shown in fig. S2, and the sequence of the was therefore protected against restriction during transplanted mycoplasma clone sMmYCp235-1 the transplantation from a native donor cell (10).
has been submitted to GenBank (accession However, the bacterial genomes grown in yeast The J. Craig Venter Institute, 9704 Medical Center Drive, are unmethylated and so are not protected from Synthetic genome assembly strategy. The Rockville, MD 20850, USA. 2The J. Craig Venter Institute, 10355Science Center Drive, San Diego, CA 92121, USA.
the single restriction system of the recipient cell.
designed cassettes were generally 1080 bp with We overcame this restriction barrier by methylat- 80-bp overlaps to adjacent cassettes (11). They *To whom correspondence should be addressed. E-mail:[email protected] ing the donor DNA with purified methylases or were all produced by assembly of chemically 2 JULY 2010 VOL 329 SCIENCE www.sciencemag.org synthesized oligonucleotides by Blue Heron coli (11). Plasmid DNA was then isolated from above to produce 100-kb assembly intermediates (Bothell, Washington). Each cassette was individ- individual E. coli clones and digested to screen for (11). Our results indicated that these products ually synthesized and sequence-verified by the cells containing a vector with an assembled 10-kb cannot be stably maintained in E. coli, so manufacturer. To aid in the building process, DNA insert. One successful 10-kb assembly is repre- recombined DNA had to be extracted from yeast.
cassettes and assembly intermediates were de- sented (Fig. 2A). In general, at least one 10-kb Multiplex polymerase chain reaction (PCR) was signed to contain Not I restriction sites at their assembled fragment could be obtained by performed on selected yeast clones (fig. S3 and termini and recombined in the presence of vector screening 10 yeast clones. However, the rate of table S2). Because every 10-kb assembly elements to allow for growth and selection in yeast success varied from 10 to 100%. All of the first- intermediate was represented by a primer pair in (7, 11). A hierarchical strategy was designed to stage intermediates were sequenced. Nineteen out this analysis, the presence of all amplicons would assemble the genome in three stages by of 111 assemblies contained errors. Alternate suggest an assembled 100-kb intermediate. In transformation and homologous recombination in clones were selected, sequence-verified, and general, 25% or more of the clones screened yeast from 1078 1-kb cassettes (Fig. 1) (12, 13).
moved on to the next assembly stage (11).
contained all of the amplicons expected for a Assembly of 10-kb synthetic intermediates.
Assembly of 100-kb synthetic intermediates.
complete assembly. One of these clones was In the first stage, cassettes and a vector were The pooled 10-kb assemblies and their respective selected for further screening. Circular plasmid recombined in yeast and transferred to Escherichia cloning vectors were transformed into yeast as DNA was extracted and sized on an agarose gelalongside a supercoiled marker. Successful second-stage assemblies with the vector sequence are 105 Elements for yeast propagation
kb in length (Fig. 2B). When all amplicons were and genome transplantation
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 on February 15, 2011 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), 1,080 bp cassettes (1,078) circular plasmids the size of our second-stage assemblies could be isolated from yeast sphero- 10,080 bp assemblies (109) plasts after an alkaline-lysis procedure. To further purify the 11 assembly intermediates, they were treated with exonuclease and passed through an 100,000 bp assemblies (11) 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 1 mg of each assembly per 400 ml of yeast culture ( 1011 cells).
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 11 circular assembly inter-mediates, 200 ng samples of each assemblywere pooled and mixed with molten agarose. As the agarose solidifies, the fibers thread through and topologically "trap" circular DNA (15).
Fig. 1. The assembly of a synthetic M. mycoides genome in yeast. A synthetic M. mycoides genome Untrapped linear DNA can then be separated was assembled from 1078 overlapping DNA cassettes in three steps. In the first step, 1080-bp out of the agarose plug by electrophoresis, thus cassettes (orange arrows), produced from overlapping synthetic oligonucleotides, were recombined enriching for the trapped circular molecules. The in sets of 10 to produce 109 10-kb assemblies (blue arrows). These were then recombined in sets of 11 circular assembly intermediates were digested 10 to produce 11 100-kb assemblies (green arrows). In the final stage of assembly, these 11 with Not I so that the inserts could be released.
fragments were recombined into the complete genome (red circle). With the exception of two Subsequently, the fragments were extracted from constructs that were enzymatically pieced together in vitro (27) (white arrows), assemblies were the agarose plug, analyzed by FIGE (Fig. 2D), carried out by in vivo homologous recombination in yeast. Major variations from the natural genome and transformed into yeast spheroplasts (11). In are shown as yellow circles. These include four watermarked regions (WM1 to WM4), a 4-kb region this third and final stage of assembly, an addi- that was intentionally deleted (94D), and elements for growth in yeast and genome transplantation.
tional vector sequence was not required because In addition, there are 20 locations with nucleotide polymorphisms (asterisks). Coordinates of thegenome are relative to the first nucleotide of the natural the yeast cloning elements were already present 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 in assembly 811-900.
1 and 800-810 were unnecessary and removed from the assembly strategy (11). Cassette 2 overlaps To screen for a complete genome, multiplex cassette 1104, and cassette 799 overlaps cassette 811.
PCR was carried out with 11 primer pairs, www.sciencemag.org SCIENCE VOL 329 2 JULY 2010 designed to span each of the 11 100-kb assembly frameshift in dnaA, an essential gene for chromo- produce the sMmYCp235 yeast strain. The dnaA- junctions (table S3). Of 48 colonies screened, somal replication. We were previously unaware of mutated genome differs by only one nucleotide DNA extracted from one clone (sMmYCp235) this mutation. By using a semisynthetic genome from the synthetic genome in sMmYCp235. This produced all 11 amplicons. PCR of the wild-type construction strategy, we pinpointed 811-900 as genome served as a negative control in our positive control (YCpMmyc1.1) produced an the source for failed synthetic transplantation transplantation experiments. The dnaA mutation indistinguishable set of 11 amplicons (Fig. 3A).
experiments. Thus, we began to reassemble an was also repaired at the 811-900 level by genome To further demonstrate the complete assembly error-free 811-900 assembly, which was used to engineering in yeast (17). A repaired 811-900 of a synthetic M. mycoides genome, intact DNAwas isolated from yeast in agarose plugs and Fig. 2. Analysis of the subjected to two restriction analyses: Asc I and BssH II (11). Because these restriction sites are (A) Not I and Sbf I double present in three of the four watermark sequences, restriction digestion anal- this choice of digestion produces restriction pat- ysis of assembly 341-350 terns that are distinct from that of the natural purified from E. coli.
M. mycoides genome (Figs. 1 and 3B). The These restriction enzymes sMmYCp235 clone produced the restriction release the vector frag-ments (5.5 and 3.4 kb) pattern expected for a completely assembled syn- from the 10-kb insert.
thetic genome (Fig. 3C).
Insert DNA was separated Synthetic genome transplantation. Additional from the vector DNA on agarose plugs used in the gel analysis above (Fig.
a 0.8% E-gel (Invitrogen).
3C) were also used in genome transplantation ex- M indicates the 1-kb DNA periments (11). Intact synthetic M. mycoides ge- ladder (New England nomes from the sMmYCp235 yeast clone were Biolabs; NEB). (B) Analy- transplanted into restriction-minus M. capricolum sis of assembly 501-600 recipient cells, as described (8). Results were purified from yeast. The scored by selecting for growth of blue colonies 105-kb circles (100-kb on SP4 medium containing tetracycline and X-gal insert plus 5-kb vector) on February 15, 2011 at 37°C. Genomes isolated from this yeast clone were separated from the produced 5 to 15 tetracycline-resistant blue colo- linear yeast chromosomal nies per agarose plug, a number comparable to that DNA on a 1% agarose Expected Size (kb) produced by the YCpMmyc1.1 control. Recovery gel by applying 4.5 V/cm of colonies in all transplantation experiments was for 3 hours. S indicates the BAC-Tracker supercoiled DNA ladder (Epicentre). (C) Not I restriction digestion dependent on the presence of both M. capricolum analysis of the 11 100-kb assemblies purified from yeast. These DNA fragments were analyzed by FIGE recipient cells and an M. mycoides genome.
on a 1% agarose gel. The expected insert size for each assembly is indicated. l indicates the lambda Semisynthetic genome assembly and trans- ladder (NEB). (D) Analysis of the 11 pooled assemblies shown in (C) following topological trapping of the plantation. To aid in testing the functionality of circular DNA and Not I digestion. One-fortieth of the DNA used to transform yeast is represented.
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 11 overlap- ping natural 100-kb assemblies in yeast by using a previously described method (16). In 11 parallel reactions, yeast cells were cotransformed with frag- mented 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 Fragment # and size (kb)
could be recombined, the PCR-amplified vectors were produced via primers with the same 40-bp (1) 685 (2) 233 (3) 160 overlaps used to clone the 100-kb synthetic as- (6) 533 (7) 233 (8) 152 semblies. The semisynthetic genomes that were constructed contained between 2 and 10 of the 11 Fig. 3. Characterization of the synthetic genome isolated from yeast. (A) Yeast clones containing a 100-kb synthetic subassemblies (Table 1). The completely assembled synthetic genome were screened by multiplex PCR with a primer set that produces 11 production of viable colonies produced after trans- amplicons; one at each of the 11 assembly junctions. Yeast clone sMmYCp235 (235) produced the 11 PCR plantation confirmed that the synthetic fraction of products expected for a complete genome assembly. For comparison, the natural genome extracted from each genome contained no lethal mutations. Only yeast (WT, wild type) was also analyzed. PCR products were separated on a 2% E-gel (Invitrogen). L indicates one of the 100-kb subassemblies, 811-900, was 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 Initially, an error-containing 811-820 clone were isolated from yeast in agarose plugs. In addition, DNA was purified from the host strain alone (H).
was used to produce a synthetic genome that did Agarose plugs were digested with Asc I or BssH II, and fragments were separated by clamped homogeneous not transplant. This was expected because the electrical field (CHEF) gel electrophoresis. Restriction fragments corresponding to the correct sizes are error was a single–base pair deletion that creates a indicated by the fragment numbers shown in (B).
2 JULY 2010 VOL 329 SCIENCE www.sciencemag.org assembly was used in a final-stage assembly to A single transplant originating from the was a complete replacement of the M. capricolum produce a yeast clone with a repaired genome.
sMmYCp235 synthetic genome was sequenced.
genome by our synthetic genome during the trans- This yeast clone is named sMmYCP142 and We refer to this strain as M. mycoides JCVI- plant process.
could be transplanted. A complete list of ge- syn1.0. The sequence matched the intended The cells with only the synthetic genome are nomes that have been assembled from 11 pieces design with the exception of the known poly- self-replicating and capable of logarithmic growth.
and successfully transplanted is provided in morphisms, eight new single-nucleotide poly- Scanning and transmission electron micrographs morphisms, an E. coli transposon insertion, and (EMs) of M. mycoides JCVI-syn1.0 cells show Characterization of the synthetic transplants.
an 85-bp duplication (table S1). The transposon small, ovoid cells surrounded by cytoplasmic mem- To rapidly distinguish the synthetic transplants insertion exactly matches the size and sequence branes (Fig. 5, C to F). Proteomic analysis of M.
from M. capricolum or natural M. mycoides, two of IS1, a transposon in E. coli. It is likely that IS1 mycoides JCVI-syn1.0 and the wild-type control analyses were performed. First, four primer pairs infected the 10-kb subassembly following its trans- (YCpMmyc1.1) by two-dimensional gel electro- that are specific to each of the four watermarks fer to E. coli. The IS1 insert is flanked by direct phoresis revealed almost identical patterns of were designed such that they produce four repeats of M. mycoides sequence, suggesting that it protein spots (fig. S4) that differed from those pre- amplicons in a single multiplex PCR reaction was inserted by a transposition mechanism. The viously reported for M. capricolum (10). Fourteen (table S4). All four amplicons were produced by 85-bp duplication is a result of a nonhomologous genes are deleted or disrupted in the M. mycoides transplants generated from sMmYCp235, but not end joining event, which was not detected in our JCVI-syn1.0 genome; however, the rate of appear- YCpMmyc1.1 (Fig. 4A). Second, the gel analysis sequence analysis at the 10-kb stage. These two ance of colonies on agar plates and the colony with Asc I and BssH II, described above (Fig.
insertions disrupt two genes that are evidently non- morphology are similar (compare Fig. 5, A and B).
3C), was performed. The restriction pattern ob- essential. We did not find any sequences in the We did observe slight differences in the growth tained was consistent with a transplant produced synthetic genome that could be identified as be- rates in a color-changing unit assay, with the JCVI- from a synthetic M. mycoides genome (Fig. 4B).
longing to M. capricolum. This indicates that there syn1.0 transplants growing slightly faster than theMmcyYCp1.1 control strain (fig. S6).
Discussion. In 1995, the quality standard for Table 1. Genomes that have been assembled from 11 pieces and successfully transplanted.
sequencing was considered to be one error in Assembly 2-100, 1; assembly 101-200, 2; assembly 201-300, 3; assembly 301-400, 4; assembly 10,000 bp, and the sequencing of a microbial ge- 401-500, 5; assembly 501-600, 6; assembly 601-700, 7; assembly 701-799, 8; assembly 811-900, nome required months. Today, the accuracy is sub- 9; assembly 901-1000, 10; assembly 1001-1104, 11. WM, watermarked assembly.
stantially higher. Genome coverage of 30 to 50× is on February 15, 2011 Synthetic fragments not unusual, and sequencing only requires a few days. However, obtaining an error-free genome thatcould be transplanted into a recipient cell to create a Reconstituted natural genome new cell controlled only by the synthetic genome 2/11 semisynthetic genome was complicated and required many quality-control with one watermark steps. Our success was thwarted for many weeks by 8/11 semisynthetic genome a single–base pair deletion in the essential gene without watermarks dnaA. One wrong base out of more than 1 million 9/11 semisynthetic genome 1–4, 6–8, 10–11 in an essential gene rendered the genome inactive, without watermarks whereas major genome insertions and deletions in 9/11 semisynthetic genome 1, 2 WM, 3 WM, 4, 6, 7 WM, 8, 10–11 nonessential parts of the genome had no observable with three watermarks effect on viability. The demonstration that our 10/11 semisynthetic genome 1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 10–11 synthetic genome gives rise to transplants with the with three watermarks characteristics of M. mycoides cells implies that the 11/11 synthetic genome, 1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 9–11 DNA sequence on which it is based is accurate 811-820 correction of dnaA enough to specify a living cell with the appropriate 11/11 synthetic genome, 1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 9–11 811-900 correction of dnaA Our synthetic genomic approach stands in sharp contrast to various other approaches to genome en-gineering that modify natural genomes by introduc- Fig. 4. Characterization of ing multiple insertions, substitutions, or deletions the transplants. (A) Trans- (18–22). This work provides a proof of principle plants containing a synthetic for producing cells based on computer-designed genome were screened by mul- genome sequences. DNA sequencing of a cel- tiplex PCR with a primer set lular genome allows storage of the genetic in- that produces four amplicons, structions for life as a digital file. The synthetic one internal to each of the genome described here has only limited modifi- four watermarks. One trans- cations from the naturally occurring M. mycoides plant (syn1.0) originating from genome. However, the approach we have de- yeast clone sMmYCp235 was veloped should be applicable to the synthesis and analyzed alongside a natural, transplantation of more novel genomes as genome nonsynthetic genome (WT) transplanted out of yeast. The transplant containing the synthetic genome produced the design progresses (23).
We refer to such a cell controlled by a genome four PCR products, whereas the WT genome did not produce assembled from chemically synthesized pieces of any. PCR products were separated on a 2% E-gel (Invitrogen).
(B) Natural (WT) and synthetic (syn1.0) M. mycoides genomes "synthetic cell," even though the cyto- were isolated from plasm of the recipient cell is not synthetic. Pheno- 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 typic effects of the recipient cytoplasm are diluted correct sizes are indicated by the fragment numbers shown in Fig. 3B.
with protein turnover and as cells carrying only the www.sciencemag.org SCIENCE VOL 329 2 JULY 2010 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 cellson SP4 agar plates con- taining X-gal. Three daysafter plating, the JCVI- syn1.0 colonies are bluebecause the cells containthe lacZ gene and express b-galactosidase, whichconverts the X-gal to ablue compound (A). TheWT cells do not containlacZ and remain white (B). Both cell types havethe fried egg colony mor-phology characteristic of most mycoplasmas. EMs were made of the JCVI-syn1.0 isolateusing two methods. (C) For scanning EM, samples were postfixed in osmium tetroxide,dehydrated and critical point dried with CO2, and visualized with a Hitachi SU6600 SEMat 2.0 keV. (D) Negatively stained transmission EMs of dividing cells with 1% uranyl acetate on pure carbon substrate visualized using JEOL 1200EX CTEM at 80 keV. Toexamine cell morphology, we compared uranyl acetate–stained EMs of M. mycoidesJCVI-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 on February 15, 2011 general appearance. EMs were provided by T. Deerinck and M. Ellisman of the National Center for Microscopy and Imaging Research at the University ofCalifornia at San Diego.
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of this work (25, 26). As synthetic genomic ap- 23. A. S. Khalil, J. J. Collins, Nat. Rev. Genet. 11, 367 (2010).
plications expand, we anticipate that this work will 24. A mycoplasma cell, with a mass of about 10−13 g, contains fewer than 106 molecules of protein. (If it 9 April 2010; accepted 13 May 2010 continue to raise philosophical issues that have contains 20% protein, this is equivalent to 2 × 10−14 g Published online 20 May 2010; broad societal and ethical implications. We en- of protein per cell. At a molecular mass of 120 daltons courage the continued discourse.
per amino acid residue, each cell contains (2 × 10−14)/ Include this information when citing this paper.
2 JULY 2010 VOL 329 SCIENCE www.sciencemag.org

Source: http://www.grid.unep.ch/FP2011/step1/pdf/028_syntheticBiology_references.pdf/028_Gibson_2010.pdf