Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System
Author(s)
Shehata, Laila; Kuldell, Natalie; Olshefsky, Audrey E.
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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 phenotype observed in the T541S mutant strain may arise because the serine sidechain is chemically similar but physically smaller than the threonine sidechain. This may minimally change the protein’s local structure, but may be less sterically constrained when compared to threonine, resulting in a higher probability of a phosphotransfer event. Our initial success pairing synthetic biology and site-directed mutagenesis to optimize the bacterial photography system’s performance encourages us to imagine further improvements to the performance of this and other synthetic systems, especially those based on 2CS signaling.
Date issued
2016-01Department
Massachusetts Institute of Technology. Department of Biological EngineeringJournal
PLOS ONE
Publisher
Public Library of Science
Citation
Olshefsky, Audrey, Laila Shehata, and Natalie Kuldell. “Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.” Edited by Dirk-Jan Scheffers. PLoS ONE 11, no. 1 (January 22, 2016): e0147494.
Version: Final published version
ISSN
1932-6203