Journal
ACS SYNTHETIC BIOLOGY
Volume 4, Issue 11, Pages 1244-1253Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.5b00133
Keywords
recombineering; Illumina sequencing; directed evolution; genome-wide expression library; genotype-phenotype mapping
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Funding
- Office of Science (BER), U.S. Department of Energy [DE-SC0008812]
- U.S. Department of Energy (DOE) [DE-SC0008812] Funding Source: U.S. Department of Energy (DOE)
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The reliable engineering of biological systems requires quantitative mapping of predictable and context-independent expression over a broad range of protein expression levels. However, current techniques for modifying expression levels are cumbersome and are not amenable to high-throughput approaches. Here we present major improvements to current techniques through the design and construction of E. coli genome-wide libraries using synthetic DNA cassettes that can tune expression over a similar to 10(4) range. The cassettes also contain molecular barcodes that are optimized for next-generation sequencing, enabling rapid and quantitative tracking of alleles that have the highest fitness advantage. We show these libraries can be used to determine which genes and expression levels confer greater fitness to E. coli under different growth conditions.
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