期刊
ACS SYNTHETIC BIOLOGY
卷 8, 期 4, 页码 655-660出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.8b00515
关键词
Synechocystis sp. PCC6803; T7 RNA polymerase/T7 promoter circuit; orthogonal promoter; nickel inducible promoter
资金
- National Science Foundation [MCB-1331151]
- Carnegie Institution for Science
To develop tightly regulated orthogonal gene expression circuits in the photoautotrophic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we designed a circuit in which a native inducible promoter drives the expression of phage T7 RNA polymerase (T7RNAP). T7RNAP, in turn, specifically recognizes the T7 promoter that is designed to drive GFP expression. In Syn6803, this T7RNAP/T7promoter-GFP circuit produces high, GFP fluorescence, which was further enhanced by using mutant T7 promoters. We also tested two orthogonal inducible promoters, Trc10 and L03, but these promoters drive T7RNAP to levels that are toxic in E. coli. Introduction of a protein degradation tag alleviated this problem. However, in Syn6803, these circuits did not function successfully. This highlights the underappreciated fact that similar circuits work with varying efficiencies in different chassis organisms. This lays the groundwork for developing new orthogonally controlled phage RNA polymerase-dependent expression systems in Syn6803.
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