Precise Manipulation of Chromosomes in Vivo Enables Genome-wide Codon Replacement

Overview

Journal Science

Specialty Science

Date 2011 Jul 19

PMID 21764749

Citations 246

Authors

Farren J Isaacs

Peter A Carr

Harris H Wang

Marc J Lajoie

Bram Sterling

Laurens Kraal

Andrew C Tolonen

Tara A Gianoulis

Daniel B Goodman

Nikos B Reppas

Christopher J Emig

Duhee Bang

Samuel J Hwang

Michael C Jewett

Joseph M Jacobson

George M Church

Affiliations

Soon will be listed here.

Abstract

We present genome engineering technologies that are capable of fundamentally reengineering genomes from the nucleotide to the megabase scale. We used multiplex automated genome engineering (MAGE) to site-specifically replace all 314 TAG stop codons with synonymous TAA codons in parallel across 32 Escherichia coli strains. This approach allowed us to measure individual recombination frequencies, confirm viability for each modification, and identify associated phenotypes. We developed hierarchical conjugative assembly genome engineering (CAGE) to merge these sets of codon modifications into genomes with 80 precise changes, which demonstrate that these synonymous codon substitutions can be combined into higher-order strains without synthetic lethal effects. Our methods treat the chromosome as both an editable and an evolvable template, permitting the exploration of vast genetic landscapes.

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References

Ambrogelly A, Palioura S, Soll D . Natural expansion of the genetic code. Nat Chem Biol. 2006; 3(1):29-35. DOI: 10.1038/nchembio847. View

Cha R, Zarbl H, Keohavong P, Thilly W . Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl. 1992; 2(1):14-20. DOI: 10.1101/gr.2.1.14. View

Jacinto-Loeza E, Vivanco-Dominguez S, Guarneros G, Hernandez-Sanchez J . Minigene-like inhibition of protein synthesis mediated by hungry codons near the start codon. Nucleic Acids Res. 2008; 36(13):4233-41. PMC: 2490762. DOI: 10.1093/nar/gkn395. View

Chan L, Kosuri S, Endy D . Refactoring bacteriophage T7. Mol Syst Biol. 2006; 1:2005.0018. PMC: 1681472. DOI: 10.1038/msb4100025. View

Coleman J, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S . Virus attenuation by genome-scale changes in codon pair bias. Science. 2008; 320(5884):1784-7. PMC: 2754401. DOI: 10.1126/science.1155761. View

Warming S, Costantino N, Court D, Jenkins N, Copeland N . Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 2005; 33(4):e36. PMC: 549575. DOI: 10.1093/nar/gni035. View

Devito J . Recombineering with tolC as a selectable/counter-selectable marker: remodeling the rRNA operons of Escherichia coli. Nucleic Acids Res. 2007; 36(1):e4. PMC: 2248734. DOI: 10.1093/nar/gkm1084. View

Posfai G, Plunkett 3rd G, Feher T, Frisch D, Keil G, Umenhoffer K . Emergent properties of reduced-genome Escherichia coli. Science. 2006; 312(5776):1044-6. DOI: 10.1126/science.1126439. View

Carr P, Park J, Lee Y, Yu T, Zhang S, Jacobson J . Protein-mediated error correction for de novo DNA synthesis. Nucleic Acids Res. 2004; 32(20):e162. PMC: 534640. DOI: 10.1093/nar/gnh160. View

10.

Oppenheim A, Rattray A, Bubunenko M, Thomason L, Court D . In vivo recombineering of bacteriophage lambda by PCR fragments and single-strand oligonucleotides. Virology. 2004; 319(2):185-9. DOI: 10.1016/j.virol.2003.11.007. View

11.

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 U S A. 2005; 102(44):15971-6. PMC: 1276048. DOI: 10.1073/pnas.0503868102. View

12.

Wang H, Isaacs F, Carr P, Sun Z, Xu G, Forest C . Programming cells by multiplex genome engineering and accelerated evolution. Nature. 2009; 460(7257):894-898. PMC: 4590770. DOI: 10.1038/nature08187. View

13.

Ochman H, Lawrence J, Groisman E . Lateral gene transfer and the nature of bacterial innovation. Nature. 2000; 405(6784):299-304. DOI: 10.1038/35012500. View

14.

Li H, Durbin R . Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14):1754-60. PMC: 2705234. DOI: 10.1093/bioinformatics/btp324. View

15.

Gibson D, Glass J, Lartigue C, Noskov V, Chuang R, Algire M . Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010; 329(5987):52-6. DOI: 10.1126/science.1190719. View

16.

Hemm M, Paul B, Miranda-Rios J, Zhang A, Soltanzad N, Storz G . Small stress response proteins in Escherichia coli: proteins missed by classical proteomic studies. J Bacteriol. 2009; 192(1):46-58. PMC: 2798279. DOI: 10.1128/JB.00872-09. View

17.

Young T, Schultz P . Beyond the canonical 20 amino acids: expanding the genetic lexicon. J Biol Chem. 2010; 285(15):11039-44. PMC: 2856976. DOI: 10.1074/jbc.R109.091306. View

18.

Carr P, Church G . Genome engineering. Nat Biotechnol. 2009; 27(12):1151-62. DOI: 10.1038/nbt.1590. View

19.

Mukai T, Hayashi A, Iraha F, Sato A, Ohtake K, Yokoyama S . Codon reassignment in the Escherichia coli genetic code. Nucleic Acids Res. 2010; 38(22):8188-95. PMC: 3001078. DOI: 10.1093/nar/gkq707. View

20.

Schaaper R, Dunn R . Spectra of spontaneous mutations in Escherichia coli strains defective in mismatch correction: the nature of in vivo DNA replication errors. Proc Natl Acad Sci U S A. 1987; 84(17):6220-4. PMC: 299042. DOI: 10.1073/pnas.84.17.6220. View