Zhang, Chao
(2022)
Engineering And Application of Bacterial Two-Component Systems.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
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Abstract
Two-component system (TCS) is fundamental to the signal sensing in bacteria which responds to a wide array of input signals. A canonical TCS consists of a histidine kinase (HK) which senses and transmits signals and a response regulator (RR) which triggers downstream responses. The functions and the interplay between these modules remain not well characterized. Here in this work, a synthetic biology approach was taken to engineer and characterize chimeric HKs in biomarker sensing and interrogating the relationship between multiple players involved in in the regulation of the cell cycle of Caulobacter crescentus.
Here, in chapter 2, a combination of synthetic biology and biochemistry approaches were taken to elucidate the role of scaffold PodJ in regulating the activity switch of the cell fate determining bifunctional kinase PleC from Caulobacter crescentus. By constructing a PleC-CcaS chimera, the key players were isolated, reconstituted and studied in E. coli. The results showed the PodJ long to short form truncation corresponds to PleC’s activity switch. In vitro biochemistry showed PodJ modulates PleC’s activity switch in a liquid-liquid phase separation (LLPS) dependent manner. This combined effect from allostery and phase separation on the modulation of activities urges us to reinspect and redefine the basic biochemical events in bacteria.
In chapter 3, a chimeric histidine kinase for indole-3-aldehyde (I3A) sensing was engineered. The sensor displayed high dynamic range, high sensitivity, and high specificity towards a library of biologically relevant indole metabolites, which holds the potential to be used as a tool for non-invasive biomarker detection and reporting in the gut.
In chapter 4, I explored the rules to engineer tandem sensors. Specifically, I’ve engineered a narrow spectrum LOV-CcaS sensor for blue light. This narrow spectrum profile is likely linked to the FRET and allosteric effects between two sensors. Truncation of the inter-sensor coiled-coil linker modulates the dynamics. This work paves the road for using tandem sensors with signal processing capability.
Overall, I explored the potential of using single and tandem sensor for their applications in biomarker detection and interpretating the interplay between scaffold and client with future directions discussed in each chapter.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
24 February 2022 |
| Date Type: |
Publication |
| Defense Date: |
26 October 2021 |
| Approval Date: |
24 February 2022 |
| Submission Date: |
29 October 2021 |
| Access Restriction: |
2 year -- Restrict access to University of Pittsburgh for a period of 2 years. |
| Number of Pages: |
189 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Chemistry |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
chimeric sensor engineering in bacteria |
| Date Deposited: |
24 Feb 2022 15:30 |
| Last Modified: |
24 Feb 2022 15:30 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/41894 |
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