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Programming biological operating systems: genome design, assembly and activation

Abstract

The DNA technologies developed over the past 20 years for reading and writing the genetic code converged when the first synthetic cell was created 4 years ago. An outcome of this work has been an extraordinary set of tools for synthesizing, assembling, engineering and transplanting whole bacterial genomes. Technical progress, options and applications for bacterial genome design, assembly and activation are discussed.

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Figure 1: Moving life into the digital world and back.
Figure 2: Options for genome assembly.
Figure 3: A synthetic genome activated in a cell-free environment will give the same outcome as a genome activated through genome transplantation.
Figure 4: A futuristic vision for designing synthetic organisms on demand.

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References

  1. Gibson, D.G. et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329, 52–56 (2010).

    Article  CAS  Google Scholar 

  2. Gibson, D.G. Oligonucleotide assembly in yeast to produce synthetic DNA fragments. Methods Mol. Biol. 852, 11–21 (2012).

    Article  CAS  Google Scholar 

  3. Gibson, D.G. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 498, 349–361 (2011).

    Article  CAS  Google Scholar 

  4. Gibson, D.G. Gene and genome construction in yeast. Curr. Protoc. Mol. Biol. 94, 3.22 (2011).

    Google Scholar 

  5. Gibson, D.G. Synthesis of DNA fragments in yeast by one-step assembly of overlapping oligonucleotides. Nucleic Acids Res. 37, 6984–6990 (2009).

    Article  CAS  Google Scholar 

  6. Gibson, D.G. et al. Complete chemical synthesis, assembly, and cloning of a Mycoplasma genitalium genome. Science 319, 1215–1220 (2008).

    Article  CAS  Google Scholar 

  7. Gibson, D.G. et al. One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome. Proc. Natl. Acad. Sci. USA 105, 20404–20409 (2008).

    Article  CAS  Google Scholar 

  8. Gibson, D.G., Smith, H.O., Hutchison, C.A. III., Venter, J.C. & Merryman, C. Chemical synthesis of the mouse mitochondrial genome. Nat. Methods 7, 901–903 (2010).

    Article  CAS  Google Scholar 

  9. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    Article  CAS  Google Scholar 

  10. Benders, G.A. et al. Cloning whole bacterial genomes in yeast. Nucleic Acids Res. 38, 2558–2569 (2010).

    Article  CAS  Google Scholar 

  11. Lartigue, C. et al. Genome transplantation in bacteria: changing one species to another. Science 317, 632–638 (2007).

    Article  CAS  Google Scholar 

  12. Lartigue, C. et al. Creating bacterial strains from genomes that have been cloned and engineered in yeast. Science 325, 1693–1696 (2009).

    Article  CAS  Google Scholar 

  13. Venter, J.C. Life at the Speed of Light: From the Double Helix to the Dawn of Digital Life (Viking, 2013).

  14. Ma, S., Tang, N. & Tian, J. DNA synthesis, assembly and applications in synthetic biology. Curr. Opin. Chem. Biol. 16, 260–267 (2012).

    Article  CAS  Google Scholar 

  15. Ellis, T., Adie, T. & Baldwin, G.S. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr. Biol. (Camb.) 3, 109–118 (2011).

    Article  CAS  Google Scholar 

  16. Itaya, M., Tsuge, K., Koizumi, M. & Fujita, K. Combining two genomes in one cell: stable cloning of the Synechocystis PCC6803 genome in the Bacillus subtilis 168 genome. Proc. Natl. Acad. Sci. USA 102, 15971–15976 (2005).

    Article  CAS  Google Scholar 

  17. Smailus, D.E., Warren, R.L. & Holt, R.A. Constructing large DNA segments by iterative clone recombination. Syst. Synth. Biol. 1, 139–144 (2007).

    Article  Google Scholar 

  18. Ma, H., Kunes, S., Schatz, P.J. & Botstein, D. Plasmid construction by homologous recombination in yeast. Gene 58, 201–216 (1987).

    Article  CAS  Google Scholar 

  19. Tagwerker, C. et al. Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast. Nucleic Acids Res. 40, 10375–10383 (2012).

    Article  CAS  Google Scholar 

  20. Karas, B.J., Tagwerker, C., Yonemoto, I.T., Hutchison, C.A. III. & Smith, H.O. Cloning the Acholeplasma laidlawii PG-8A genome in Saccharomyces cerevisiae as a yeast centromeric plasmid. ACS Synth. Biol. 1, 22–28 (2012).

    Article  CAS  Google Scholar 

  21. Karas, B.J. et al. Direct transfer of whole genomes from bacteria to yeast. Nat. Methods 10, 410–412 (2013).

    Article  CAS  Google Scholar 

  22. Schwartz, D.C. & Cantor, C.R. Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37, 67–75 (1984).

    Article  CAS  Google Scholar 

  23. Noskov, V.N. et al. Assembly of large, high G+C bacterial DNA fragments in yeast. ACS Synth. Biol. 1, 267–273 (2012).

    Article  CAS  Google Scholar 

  24. Kouprina, N. & Larionov, V. Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae. Nat. Protoc. 3, 371–377 (2008).

    Article  CAS  Google Scholar 

  25. Kornberg, R.D. Eukaryotic transcriptional control. Trends Cell Biol. 9, M46–M49 (1999).

    Article  CAS  Google Scholar 

  26. Kozak, M. Initiation of translation in prokaryotes and eukaryotes. Gene 234, 187–208 (1999).

    Article  CAS  Google Scholar 

  27. Marschall, P., Malik, N. & Larin, Z. Transfer of YACs up to 2.3 Mb intact into human cells with polyethylenimine. Gene Ther. 6, 1634–1637 (1999).

    Article  CAS  Google Scholar 

  28. van Brabant, A.J., Buchanan, C.D., Charboneau, E., Fangman, W.L. & Brewer, B.J. An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint. Mol. Cell 7, 705–713 (2001).

    Article  CAS  Google Scholar 

  29. Carr, P.A. & Church, G.M. Genome engineering. Nat. Biotechnol. 27, 1151–1162 (2009).

    Article  CAS  Google Scholar 

  30. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).

    Article  CAS  Google Scholar 

  31. Isaacs, F.J. et al. Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333, 348–353 (2011).

    Article  CAS  Google Scholar 

  32. Ellis, H.M., Yu, D., DiTizio, T. & Court, D.L. High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc. Natl. Acad. Sci. USA 98, 6742–6746 (2001).

    Article  CAS  Google Scholar 

  33. Dymond, J.S. et al. Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature 477, 471–476 (2011).

    Article  CAS  Google Scholar 

  34. Gaj, T., Gersbach, C.A. & Barbas, C.F. III. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol. 31, 397–405 (2013).

    Article  CAS  Google Scholar 

  35. Gurdon, J.B. & Byrne, J.A. The first half-century of nuclear transplantation. Proc. Natl. Acad. Sci. USA 100, 8048–8052 (2003).

    Article  CAS  Google Scholar 

  36. Gurdon, J.B. & Wilmut, I. Nuclear transfer to eggs and oocytes. Cold Spring Harb. Perspect. Biol. 3, a002659 (2011).

    Article  Google Scholar 

  37. Forster, A.C. & Church, G.M. Towards synthesis of a minimal cell. Mol. Syst. Biol. 2, 45 (2006).

    Article  Google Scholar 

  38. Forster, A.C. & Church, G.M. Synthetic biology projects in vitro. Genome Res. 17, 1–6 (2007).

    Article  CAS  Google Scholar 

  39. Jewett, M.C. & Forster, A.C. Update on designing and building minimal cells. Curr. Opin. Biotechnol. 21, 697–703 (2010).

    Article  CAS  Google Scholar 

  40. Medema, M.H., van Raaphorst, R., Takano, E. & Breitling, R. Computational tools for the synthetic design of biochemical pathways. Nat. Rev. Microbiol. 10, 191–202 (2012).

    Article  CAS  Google Scholar 

  41. Chan, L.Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. Mol. Syst. Biol. 1, 2005.0018 (2005).

    Article  Google Scholar 

  42. Jaschke, P.R., Lieberman, E.K., Rodriguez, J., Sierra, A. & Endy, D. A fully decompressed synthetic bacteriophage oX174 genome assembled and archived in yeast. Virology 434, 278–284 (2012).

    Article  CAS  Google Scholar 

  43. Fraser, C.M. et al. The minimal gene complement of Mycoplasma genitalium. Science 270, 397–403 (1995).

    Article  CAS  Google Scholar 

  44. Karr, J.R. et al. A whole-cell computational model predicts phenotype from genotype. Cell 150, 389–401 (2012).

    Article  CAS  Google Scholar 

  45. Dormitzer, P.R. et al. Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci. Transl. Med. 5, 185ra68 (2013).

    Article  Google Scholar 

  46. Guye, P., Li, Y., Wroblewska, L., Duportet, X. & Weiss, R. Rapid, modular and reliable construction of complex mammalian gene circuits. Nucleic Acids Res. 41, e156 (2013).

    Article  Google Scholar 

  47. Temme, K., Zhao, D. & Voigt, C.A. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc. Natl. Acad. Sci. USA 109, 7085–7090 (2012).

    Article  Google Scholar 

  48. O'Neill, B.M. et al. An exogenous chloroplast genome for complex sequence manipulation in algae. Nucleic Acids Res. 40, 2782–2792 (2012).

    Article  CAS  Google Scholar 

  49. Smith, H.O., Hutchison, C.A. III., Pfannkoch, C. & Venter, J.C. Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 100, 15440–15445 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

I would like to thank past and present members of the synthetic biology groups at the J. Craig Venter Institute and Synthetic Genomics, Inc., for making all of these technologies I discuss here possible, especially C. Venter, H. Smith and C. Hutchison, who had the vision to make a synthetic cell as early as 1995. I also thank M. LaPointe, T. Richardson, J. Ward and J. Eads for their contributions to Figures 1, 2 and 4 and C. Hutchison for producing Figure 3.

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Correspondence to Daniel G Gibson.

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D.G.G. is Vice President of DNA Technologies at Synthetic Genomics, Inc., and holds employee stock shares in this company.

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Gibson, D. Programming biological operating systems: genome design, assembly and activation. Nat Methods 11, 521–526 (2014). https://doi.org/10.1038/nmeth.2894

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