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

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 ∼104 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.