Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System

PloS One
Audrey OlshefskyNatalie Kuldell

Abstract

Two-component signaling (2CS) systems enable bacterial cells to respond to changes in their local environment, often using a membrane-bound sensor protein and a cytoplasmic responder protein to regulate gene expression. Previous work has shown that Escherichia coli's natural EnvZ/OmpR 2CS could be modified to construct a light-sensing bacterial photography system. The resulting bacterial photographs, or "coliroids," rely on a phosphotransfer reaction between Cph8, a synthetic version of EnvZ that senses red light, and OmpR. Gene expression changes can be visualized through upregulation of a LacZ reporter gene by phosphorylated OmpR. Unfortunately, basal LacZ expression leads to a detectable reporter signal even when cells are grown in the light, diminishing the contrast of the coliroids. We performed site-directed mutagenesis near the phosphotransfer site of Cph8 to isolate mutants with potentially improved image contrast. Five mutants were examined, but only one of the mutants, T541S, increased the ratio of dark/light gene expression, as measured by β-galactosidase activity. The ratio changed from 2.57 fold in the starting strain to 5.59 in the T541S mutant. The ratio decreased in the four other mutant strains we examined. The...Continue Reading

References

Nov 25, 2005·Nature·Anselm LevskayaChristopher A Voigt
Jan 22, 2010·Nature·Tal DaninoJeff Hasty
May 29, 2012·Current Opinion in Chemical Biology·Laura Gardner, Alexander Deiters
Jul 17, 2014·Nature Reviews. Molecular Cell Biology·Doug Tischer, Orion D Weiner
May 17, 2015·Applied and Environmental Microbiology·Teng ChuYuanxing Zhang

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Citations

May 18, 2021·Synthetic and Systems Biotechnology·Logan R BurringtonJavin P Oza

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Methods Mentioned

BETA
Assay
phosphotransferase
phosphotranferase
biosensor

Software Mentioned

NIH
Plot Lanes
ImageJ
GENEWIZ

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