Engineering bacterial two-component sensors with sub-second response times
Abstract
The goal of this proposal is to develop a new technology to engineer living bacteria to specifically sense and rapidly report the presence of a wide variety of chemical and physical stimuli. Bacterial two-component systems (TCSs) are the largest and most diverse" family of sensors in biology. We have previously engineered TCSs to sense a wide range of optical and chemical inputs. However, due"" to their gene expression outputs, our first generation TCS sensors exhibit slow (~1 hour) response dynamics. Here, we propose to ac"celerate the response dynamics of TCS sensors by three orders of magnitude by replacing gene expression outputs with optical reporter proteins that produce signals in response to the phosphorylation state of the TCS response regulator (RR) protein. In the absence" of input, RRs are non-phosphorylated and monomeric. In the presence of input, RRs are phosphorylated and dimeric. Phosphorylation-i"nduced dimerization is fully reversible and occurs in less than one second. We will fuse RRs to fluorescent proteins capable of undergoing Forster Resonance Energy Transfer (FRET) when the RRs are dimerized and utilize FRET signal as a TCS output with sub-second k"inetics. First, we will validate this technology using a previous light-sensing TCS that we have engineered. We have chosen this TCS" as a model because it is very-well understood and can the light input signals can be controlled with millisecond time resolution us"ing standard laboratory equipment. Second, we will use this technology to engineer 20 novel TCS sensors of chemicals linked to healt""h-related human gut microbiome pathways, aquatic pollution, and other important biological processes. Once demonstrated, our FRET re"porters could be directly used in biosensing applications or swapped for other rapid reporters such as split fluorescent proteins or" split luciferase. If successful, this work will transform biosensor engineering, with numerous Navy-relevant applications. Objecti"ves: Engineer 20 Bacterial Two-Component Sensors (TCS) sensors with sub-second response times by utilizing Fluorescence Resonance Energy Transfer (FRET) to monitor phosphorylation-mediated RR dimerization in vivo. This goal will be achieved through two Specific Objectives: 1)Demonstrate sub-second response times with a TCS light sensor 2)Engineer 20 TCS sensors of biologically- and environmentally-relevant chemicals with sub-second response times
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 07, 2017
- Source ID
- N000141712642
Entities
People
- Jeffrey J Tabor
Organizations
- Office of Naval Research
- Rice University
- United States Navy