Genome-Wide Tuning of Protein Expression Levels to Rapidly Engineer Microbial Traits

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• 作者：Emily F. Freed ; James D. Winkler ; Sophie J. Weiss ; Andrew D. Garst ; Vivek K. Mutalik ; Adam P. Arkin ; Rob Knight ; Ryan T. Gill
• 刊名：ACS Synthetic Biology
• 出版年：2015
• 出版时间：November 20, 2015
• 年：2015
• 卷：4
• 期：11
• 页码：1244-1253
• 全文大小：598K
• ISSN：2161-5063

文摘

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 鈭?0⁴ 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.