Reader TTS

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

Access through your institution Subscribe to this journal Receive 12 print issues and online access $259.00 per year only $21.58 per issue Learn more Buy this article * Purchase on

SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about

institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS BUILDING GENOMES TO UNDERSTAND BIOLOGY Article Open access 02 December 2020 THE

TRANSCRIPTIONAL LANDSCAPE OF A REWRITTEN BACTERIAL GENOME REVEALS CONTROL ELEMENTS AND GENOME DESIGN PRINCIPLES Article Open access 24 May 2021 THE DESIGN AND ENGINEERING OF SYNTHETIC

GENOMES Article 06 November 2024 REFERENCES * 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  * Gibson, D.G. Oligonucleotide assembly in yeast to produce synthetic DNA fragments. _Methods Mol. Biol._ 852, 11–21 (2012). Article  CAS  Google Scholar  * Gibson, D.G.

Enzymatic assembly of overlapping DNA fragments. _Methods Enzymol._ 498, 349–361 (2011). Article  CAS  Google Scholar  * Gibson, D.G. Gene and genome construction in yeast. _Curr. Protoc.

Mol. Biol._ 94, 3.22 (2011). Google Scholar  * 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  * 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  * 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  * 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  * 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  * Benders, G.A. et al. Cloning whole bacterial genomes in yeast. _Nucleic Acids Res._ 38, 2558–2569 (2010). Article  CAS  Google Scholar  * Lartigue, C.

et al. Genome transplantation in bacteria: changing one species to another. _Science_ 317, 632–638 (2007). Article  CAS  Google Scholar  * 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  * Venter, J.C. _Life at the Speed of Light: From the Double Helix

to the Dawn of Digital Life_ (Viking, 2013). * 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  * 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  * 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  * 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  * 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  * 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  * 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  * Karas, B.J. et al.

Direct transfer of whole genomes from bacteria to yeast. _Nat. Methods_ 10, 410–412 (2013). Article  CAS  Google Scholar  * 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  * 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  * 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  * Kornberg, R.D. Eukaryotic

transcriptional control. _Trends Cell Biol._ 9, M46–M49 (1999). Article  CAS  Google Scholar  * Kozak, M. Initiation of translation in prokaryotes and eukaryotes. _Gene_ 234, 187–208 (1999).

Article  CAS  Google Scholar  * 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  * 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  * Carr, P.A. & Church, G.M. Genome engineering. _Nat. Biotechnol._ 27, 1151–1162 (2009). Article  CAS  Google

Scholar  * Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. _Nature_ 460, 894–898 (2009). Article  CAS  Google Scholar  * 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  * 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  * 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  *

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  *

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  * Gurdon, J.B. &

Wilmut, I. Nuclear transfer to eggs and oocytes. _Cold Spring Harb. Perspect. Biol._ 3, a002659 (2011). Article  Google Scholar  * Forster, A.C. & Church, G.M. Towards synthesis of a

minimal cell. _Mol. Syst. Biol._ 2, 45 (2006). Article  Google Scholar  * Forster, A.C. & Church, G.M. Synthetic biology projects _in vitro_. _Genome Res._ 17, 1–6 (2007). Article  CAS 

Google Scholar  * Jewett, M.C. & Forster, A.C. Update on designing and building minimal cells. _Curr. Opin. Biotechnol._ 21, 697–703 (2010). Article  CAS  Google Scholar  * 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  * Chan, L.Y., Kosuri, S. & Endy, D. Refactoring bacteriophage T7. _Mol. Syst. Biol._ 1, 2005.0018 (2005). Article  Google Scholar  * 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  *

Fraser, C.M. et al. The minimal gene complement of _Mycoplasma genitalium_. _Science_ 270, 397–403 (1995). Article  CAS  Google Scholar  * Karr, J.R. et al. A whole-cell computational model

predicts phenotype from genotype. _Cell_ 150, 389–401 (2012). Article  CAS  Google Scholar  * 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  * 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  * 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  * 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  * 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  Download

references 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. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * J. Craig Venter

Institute, La Jolla, California, USA Daniel G Gibson * Synthetic Genomics, Inc., La Jolla, California, USA Daniel G Gibson Authors * Daniel G Gibson View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Daniel G Gibson. ETHICS DECLARATIONS COMPETING INTERESTS D.G.G. is Vice President of DNA Technologies at

Synthetic Genomics, Inc., and holds employee stock shares in this company. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Gibson, D. Programming

biological operating systems: genome design, assembly and activation. _Nat Methods_ 11, 521–526 (2014). https://doi.org/10.1038/nmeth.2894 Download citation * Received: 11 December 2013 *

Accepted: 22 February 2014 * Published: 29 April 2014 * Issue Date: May 2014 * DOI: https://doi.org/10.1038/nmeth.2894 SHARE THIS ARTICLE Anyone you share the following link with will be

able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing

initiative