Optimizing two‐component light‐switchable systems for controlling gene expression in Escherichia coli (991.2)

Abstract

Light‐induced gene expression is a powerful tool for manipulating cells because it allows for precise spatial and temporal control of cellular behavior. A light‐sensitive two‐component system based on the Synechocystis PCC6803 phytochrome CcaS and the cognate response regulator CcaR has been previously constructed in Escherichia coli.1 The chromophore‐binding domain of CcaS performs a reversible photoconversion between a green (~535 nm) and a red (~672 nm) light absorbing form. The green form phosphorylates CcaR, which then induces expression of the cpcG2 promoter by binding to a conserved direct repeat designated the G‐box. Green light‐induced transcription from PcpcG2 was about ~4.3‐fold for this original construct.In order to engineer a constitutively expressed green/red light‐switchable two‐component system with improved fold‐inducibility, we built different ccaS expression cassettes using the constitutive promoter J23106 and RBSs with various translation initiation rates. This library was first screened against an aTc‐inducible expression system for CcaR resulting in a ~40 fold‐inducibility. After screening different ccaR expression cassettes using the constitutive promoter J23100 and RBSs with various translation initiation rates, a fold induction as high as ~80‐fold for a specific CcaS/CcaR‐expression pair could be observed.There are over 10,000 two‐component systems in nature, and discovering the design and expression rules for these systems will allow engineering of a wide variety of genetic circuits for practical applications. The increase in fold‐induction in this system makes light‐induction a more powerful method of spatiotemporal control of living cells and aids precise cellular studies in synthetic and systems biology.Grant Funding Source: Supported by an NSF award from the Division of Biological Infrastructure No. 1004853.