Abstract
We present a light-switchable gene expression system for both inducible and switchable control of gene expression at a single cell level in Escherichia coli using a previously constructed light-sensing system. The λ cI repressor gene with an LVA degradation tag was expressed under the control of the ompC promoter on the chromosome. The green fluorescent protein (GFP) gene fused to a λ repressor-repressible promoter was used as a reporter. This light-switchable system allows rapid and reversible induction or repression of expression of the target gene at any desired time. This system also ensures homogenous expression across the entire cell population. We also report the design of a miniaturized photobioreactor to be used in combination with the light-switchable gene expression system. The miniaturized photobioreactor helps to reduce unintended induction of the light receptor due to environmental disturbances and allows precise control over the duration of induction. This system would be a good tool for switchable, homogenous, strong, and highly regulatable expression of target genes over a wide range of induction times. Hence, it could be applied to study gene function, optimize metabolic pathways, and control biological systems both spatially and temporally.
Highlights
Switchable gene expression systems would aid in reversible induction or repression of target genes at any desired time and reduce pleiotropic effects caused by over-expression
Precise and temporally switchable control of bacterial gene expression in response to external stimuli is an essential tool for understanding and manipulating complex biological systems
Light-switchable Gene Expression System The major components of the light-switchable gene expression system used in this study include the strain JM1012 (MG1655/ Phosphorylated OmpR binds to the ompC promoter (PompC)::cI-LVA DenvZ) and 3 plasmids: 2 for sensing light and 1 reporter plasmid (Fig. 2)
Summary
Switchable gene expression systems would aid in reversible induction or repression of target genes at any desired time and reduce pleiotropic effects caused by over-expression. Several switchable gene expression systems have been developed based on physicochemical stimuli [5,6]. Chemical inducers such as aTC, arabinose, and IPTG are expensive and toxic; their separation from the final products requires additional downstream operations [7]. The use of thermo-regulatable promoters that can be induced by altering the temperature can cause protein aggregation, resulting in low yield of soluble proteins. This is not a cost-effective switchable induction method, as rapid shifts in temperature are difficult to achieve [8,9]
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