Research Article

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

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Science  02 Jul 2010:
Vol. 329, Issue 5987, pp. 52-56
DOI: 10.1126/science.1190719
  • Fig. 1

    The assembly of a synthetic M. mycoides genome in yeast. A synthetic M. mycoides genome was assembled from 1078 overlapping DNA cassettes in three steps. In the first step, 1080-bp cassettes (orange arrows), produced from overlapping synthetic oligonucleotides, were recombined in sets of 10 to produce 109 ~10-kb assemblies (blue arrows). These were then recombined in sets of 10 to produce 11 ~100-kb assemblies (green arrows). In the final stage of assembly, these 11 fragments 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 (WM1 to 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 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). (B) 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). (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. λ indicates the lambda ladder (NEB). (D) Analysis of the 11 pooled 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.

  • 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, 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).

  • Fig. 4

    Characterization of the transplants. (A) Transplants containing a synthetic genome were screened by multiplex PCR with a primer set that produces four amplicons, one internal to each of the four watermarks. One transplant (syn1.0) originating from yeast clone sMmYCp235 was analyzed alongside a natural, 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.

  • 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 β-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 morphology characteristic of most mycoplasmas. EMs were made of the JCVI-syn1.0 isolate using 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 SEM at 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. 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 T. Deerinck and M. Ellisman of the National Center for Microscopy and Imaging Research at the University of California at San Diego.

  • 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, watermarked assembly.

    Genome assemblySynthetic fragmentsNatural fragments
    Reconstituted natural genomeNone1–11
    2/11 semisynthetic genome with one watermark5 WM, 101–4, 6–9, 11
    8/11 semisynthetic genome without watermarks1–4, 6–8, 115, 9, 10
    9/11 semisynthetic genome without watermarks1–4, 6–8, 10–115, 9
    9/11 semisynthetic genome with three watermarks1, 2 WM, 3 WM, 4, 6, 7 WM, 8, 10–115, 9
    10/11 semisynthetic genome with three watermarks1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 10–119
    11/11 synthetic genome, 811-820 correction of dnaA1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 9–11None
    11/11 synthetic genome, 811-900 correction of dnaA1, 2 WM, 3 WM, 4, 5 WM, 6, 7 WM, 8, 9–11None

Additional Files


  • Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome
    Daniel G. Gibson, John I. Glass, Carole Lartigue, Vladimir N. Noskov, Ray-Yuan Chuang, Mikkel A. Algire, Gwynedd A. Benders, Michael G. Montague, Li Ma, Monzia M. Moodie, Chuck Merryman, Sanjay Vashee, Radha Krishnakumar, Nacyra Assad- Garcia, Cynthia Andrews-Pfannkoch, Evgeniya A. Denisova, Lei Young, Zhi-Qing Qi, Thomas H. Segall-Shapiro, Christopher H. Calvey, Prashanth P. Parmar, Clyde A. Hutchison III, Hamilton O. Smith, J. Craig Venter

    Supporting Online Material

    This supplement contains:
    Materials and Methods
    Figs. S1 to S6
    Tables S1 to S7
    References

    This file is in Adobe Acrobat PDF format.

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    From the Science Podcast: an interview with Craig Venter on the creation of a bacterial cell controlled by a chemically synthesized genome. (21 May 2010 Science Podcast)

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