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One-Step Assembly in Yeast of 25 Overlapping DNA Fragments to Form a Complete Synthetic Mycoplasma Genitalium Genome

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One-Step Assembly in Yeast of 25 Overlapping DNA Fragments to Form a Complete Synthetic Mycoplasma Genitalium Genome One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome Daniel G. Gibsona,1, Gwynedd A. Bendersb, Kevin C. Axelroda, Jayshree Zaveria, Mikkel A. Algirea, Monzia Moodiea, Michael G. Montaguea, J. Craig Ventera, Hamilton O. Smithb, and Clyde A. Hutchison IIIb,1 aThe J. Craig Venter Institute, Synthetic Biology Group, Rockville, MD 20850 and bThe J. Craig Venter Institute, Synthetic Biology Group, San Diego, CA 92121 Contributed by Clyde A. Hutchison III, October 30, 2008 (sent for review September 11, 2008) We previously reported assembly and cloning of the synthetic JCVI-1.0 genome. At the time, we wondered whether it would be Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharo- possible to assemble DNA fragments from earlier stages into a myces cerevisiae by recombination of six overlapping DNA frag- complete genome in a single step in yeast. ments to produce a 592-kb circle. Here we extend this approach by The assembly intermediates from our construction of the syn- demonstrating assembly of the synthetic genome from 25 over- thetic JCVI-1.0 genome provided a resource for exploring the limits lapping fragments in a single step. The use of yeast recombination of yeast uptake and assembly. Here we report the successful greatly simplifies the assembly of large DNA molecules from both assembly of the entire synthetic genome from 25 A-series assem- synthetic and natural fragments. blies in a single step. in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ Results ͉ ͉ yeast transformation Mycoplasma genitalium synthetic biology Assembly of the M. genitalium Genome in Yeast from 25 Overlapping DNA Fragments. Our 25 A-series assemblies comprising the entire east has long been considered a genetically tractable organism M. genitalium genome were all synthesized and cloned as bacterial Ybecause of its ability to take up and recombine DNA frag- artificial chromosomes (BACs) in Escherichia coli as described ments. More than 30 years ago, Hinnen et al. (1) reported the previously (8) (Table 1). For this study, two variants of assembly restoration of leucine biosynthesis in Saccharomyces cerevisiae by A86–89 were produced, with the goal of constructing M. genitalium - ϩ transformation of a leu2 strain to LEU2 using a method involving JCVI-1.1 and JCVI-1.9. Each variant has a vector inserted at the spheroplasts, CaCl2, and PEG. Soon after, Orr-Weaver et al. (2) same site within a nonessential gene. Each vector contains a reported mechanistic studies demonstrating that DNA molecules histidine auxotrophic marker, a centromere, and an origin of taken up during yeast transformation can integrate into yeast replication for selection and maintenance in yeast. The only dif- chromosomes through homologous recombination, and that the ference between JCVI-1.1 and JCVI-1.9 is in the additional vector ends of the linear-transforming DNA are highly recombinogenic features. All 25 of these A-series molecules were purified after and react directly with homologous chromosomal sequences, propagation in E. coli. The assemblies were released from their whereas circular plasmids carrying yeast sequences integrate by a respective BACs by cleavage with restriction enzymes (Table 1). single crossover and only at low frequency. Subsequently, yeast The presence of yeast propagation elements within one of our transformation has become an indispensable tool in yeast genetics. Yeast recombination has since been applied to the construction assemblies (A86–89) allowed us to investigate whether yeast has the of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma ability to assemble all of these 25 fragments into the M. genitalium et al. (3) constructed plasmids from two cotransformed DNA genome. Fig. 1 illustrates our ambitious question: Can a single yeast fragments containing homologous regions. In another process, cell take up all 25 of these pieces, translocate them to the nucleus, called linker-mediated assembly, any DNA sequence can be joined and then recombine them into the complete genome? to a vector DNA using short synthetic linkers that bridge the ends After digestion, each of the 25 assemblies contained at least 80 (4, 5). Similarly, four or five overlapping DNA pieces can be bp of overlapping sequence with the adjacent molecule at either end assembled and joined to vector DNA (4, 6, 7). This work demon- (Table 1). Equal amounts (Ϸ100 ng or Ϸ4 ϫ 109 genome equiva- strated that yeast cells can take up multiple pieces of DNA, and that lents) of each digested piece were pooled together without gel homologous yeast recombination is sufficiently efficient to correctly purification to assemble M. genitalium JCVI-1.1. Then Ϸ108 yeast assemble the pieces into a single recombinant molecule. spheroplasts were added to this DNA pool, and yeast transforma- The limitations of assembly methods in yeast remain unknown. tion was performed (9). This amounts to Ϸ40 M. genitalium genome We recently assembled an entire synthetic M. genitalium genome equivalents, or Ϸ1000 DNA molecules (40 ϫ 25) per yeast sphero- using a combination of in vitro enzymatic recombination in early plast. After incubation for 3 days at 30 °C on selective medium, stages and in vivo yeast recombination in the final stage to produce Ϸ800 colonies were obtained. the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to Author contributions: D.G.G., G.A.B., J.C.V., H.O.S., and C.A.H. designed research; M.A.A. make 1/8 genome Ϸ72-kb B-series assemblies, and then two B- and M.G.M. contributed new reagents/analytic tools; D.G.G., G.A.B., K.C.A., J.Z., and M.M. Ϸ performed research; D.G.G., G.A.B., H.O.S., and C.A.H. analyzed data; and D.G.G., G.A.B., series assemblies were assembled to make each of the 145-kb H.O.S., and C.A.H. wrote the paper. quarter-genome C-series assemblies. We accomplished the final Conflict of interest statement: C.A.H., H.O.S., and J.C.V. have a financial interest in Synthetic assembly in yeast using three quarter-genome fragments and a Genomics, Inc (SGI). The research reported in this paper is in the field in which SGI is fourth quarter fragment that had been cleaved by a restriction developing products. SGI has funded the research reported in the paper. enzyme to provide a site for insertion of the vector DNA. Thus, Freely available online through the PNAS open access option. some individual yeast cells have the capacity to simultaneously take 1To whom correspondence may be addressed. E-mail: [email protected] or chutchison@ up the overlapping genome and vector DNA fragments (a total of jcvi.org. six pieces) and recombine them to produce the full 592-kb circular © 2008 by The National Academy of Sciences of the USA 20404–20409 ͉ PNAS ͉ December 23, 2008 ͉ vol. 105 ͉ no. 51 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811011106 Downloaded by guest on October 2, 2021 Table 1. Summary of the 25 A-series assemblies used A Amplicon size (bp) B in constructing the M. genitalium synthetic genomes A1-4 A5-8 Set 1 Set 2 Set 3 Set 4 A9-12 Size, Restriction 5Ј overlap, 3Ј overlap, a 100 146 125 175 .....etc. Assembly bp digest bp bp b 200 250 225 275 j a c 300 348 325 375 b A1–4 23,636 BsmBI 80 80 i d 400 450 425 475 A5–8 26,166 NotI 80 80 e 500 550 525 575 JCVI-1.1 c A9–12 23,872 BsmBI 80 80 h A13–16 23,653 SalI and NotI 80 80 f 600 650 625 675 d A17–20 23,312 NotI 80 80 g 700 750 725 775 g A21–24 23,211 NotI 80 80 800 850 825 875 h f e A25–28 24,992 NotI 80 80 i 900 950 925 975 A29–32 26,257 NotI 80 209 j 1000 1050 1025 1075 A33–36 20,448 NotI 209 80 A37–41 25,706 NotI 80 257 A42–45 19,223 NotI 257 257 C Clone # A46–49 19,895 NotI 257 257 c5c4c3c2c1L c8c7c6 c9 c10 A50–53 24,390 NotI 257 80 1-j A54–57 24,789 NotI 80 80 1000bp 1-i 1-h A58–61 24,534 NotI 80 80 800bp 1-g 600bp 1-f A62–65 25,055 NotI 80 80 1-e 400bp 1-d A66–69 23,128 NotI 80 80 300bp A70–73 23,345 NotI 80 80 1-c 200bp A74–77 21,396 NotI 80 165 1-b A78–81 22,320 NotI 165 360 100bp 1-a A82–85 23,460 NotI 360 85 A86–89–1.1 variant 34,950 NotI 85 80 A86–89–1.9 variant 31,726 NotI 85 80 D Primer set Primer set A90–93 23,443 NotI 80 80 1 2 3 4 L L 1 2 3 4 A94–97 24,511 NotI 80 80 GENETICS A98–101 17,372 NotI 80 80 1000bp 800bp 600bp 400bp Analysis of 10 Clones by Multiplex PCR. We first screened for yeast 300bp cells that took up all 25 pieces. Forty primer pairs, producing amplicons ranging in size from 100 bp to 1075 bp, were designed 200bp such that they could produce 10 amplicons in each of four individual 100bp JCVI-1.1 (c4) JCVI-1.0 25 overlapping DNA fragments Fig. 2. Multiplex PCR analysis to screen for yeast cells that took up all 25 segments.
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