Balancing gene expression without library construction via a reusable sRNA pool. Issue 13 (13th June 2017)

• Record Type: Journal Article
• Authors: Ghodasara, Amar; Voigt, Christopher A.
• Is Part Of: Nucleic acids research. Volume 45:Issue 13(2017)
• Page Start: 8116
• Page End: 8127
• Publication Date: 2017-06-13
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkx530
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Balancing protein expression is critical when optimizing genetic systems. Typically, this requires library construction to vary the genetic parts controlling each gene, which can be expensive and time-consuming. Here, we develop sRNAs corresponding to 15nt 'target' sequences that can be inserted upstream of a gene. The targeted gene can be repressed from 1.6- to 87-fold by controlling sRNA expression using promoters of different strength. A pool is built where six sRNAs are placed under the control of 16 promoters that span a ∼10 3 -fold range of strengths, yielding ∼10 7 combinations. This pool can simultaneously optimize up to six genes in a system. This requires building only a single system-specific construct by placing a target sequence upstream of each gene and transforming it with the pre-built sRNA pool. The resulting library is screened and the top clone is sequenced to determine the promoter controlling each sRNA, from which the fold-repression of the genes can be inferred. The system is then rebuilt by rationally selecting parts that implement the optimal expression of each gene. We demonstrate the versatility of this approach by using the same pool to optimize a metabolic pathway (β-carotene) and genetic circuit (XNOR logic gate).