title: Site-Directed Mutagenesis to Improve Sensitivity of a Synthetic Two-Component Signaling System.
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 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.
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
authors.forename: Audrey
authors.initials: A
authors.lastname: Olshefsky
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States of America.
authors.forename: Laila
authors.initials: L
authors.lastname: Shehata
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States of America.|BioBuilder Educational Foundation, Cambridge, MA, United States of America.
authors.forename: Natalie
authors.initials: N
authors.lastname: Kuldell
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D007763:Lac Operon / Q000235:genetics / Q000502:physiology; D008027:Light; D009097:Multienzyme Complexes / Q000235:genetics / Q000502:physiology; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000502:physiology; D010781:Photography; D015398:Signal Transduction / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000502:physiology; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D015534:Trans-Activators; C112177:osmolarity response regulator proteins; C057843:envZ protein, E coli; D001616:beta-Galactosidase