Publication Date:
2011-07-19
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Isaacs, Farren J -- Carr, Peter A -- Wang, Harris H -- Lajoie, Marc J -- Sterling, Bram -- Kraal, Laurens -- Tolonen, Andrew C -- Gianoulis, Tara A -- Goodman, Daniel B -- Reppas, Nikos B -- Emig, Christopher J -- Bang, Duhee -- Hwang, Samuel J -- Jewett, Michael C -- Jacobson, Joseph M -- Church, George M -- K99 GM081450/GM/NIGMS NIH HHS/ -- R00 GM081450/GM/NIGMS NIH HHS/ -- New York, N.Y. -- Science. 2011 Jul 15;333(6040):348-53. doi: 10.1126/science.1205822.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. farren.isaacs@yale.edu〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21764749" target="_blank"〉PubMed〈/a〉
Keywords:
Chromosomes, Bacterial/*genetics
;
*Codon, Terminator
;
*Conjugation, Genetic
;
Directed Molecular Evolution
;
Escherichia coli/*genetics/growth & development/physiology
;
Genetic Engineering/*methods
;
*Genome, Bacterial
;
Genomic Instability
;
Mutagenesis, Site-Directed
;
Mutation
;
Phenotype
;
Recombination, Genetic
;
Templates, Genetic
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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