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Development of synthetic biology tools driven by membrane potential
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Delise, Marco A. (2020) Development of synthetic biology tools driven by membrane potential. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3491800~S15
Abstract
Every cell, from prokaryotic to mammalian cells, has a membrane sustaining an electrical potential difference, called membrane potential, which can provide an interface between external environment and cellular processes.
Following the discovery of an eukaryotic voltage-sensitive-domain (VSD) that changes conformation upon membrane potential shifts and that is independent from other attached domains, membrane potential reporters for mammalian cells have been obtained by coupling the VSD with fluorescent proteins.
In this work, I described the efforts of using such a VSD to develop a membrane potential sensor for prokaryotes and a voltage-sensitive protease for mammalian cells. First, as recent discoveries in Bacillus subtilis showed the tight involvement of membrane potential in biological processes such as cell-cell communication, biofilm formation and sporulation, a tool to better study prokaryotic membrane potential was needed. The Prokaryotic ASAP1-based Potential Sensor (PAPS) constructs were obtained by codon-optimizing the eukaryotic VSD for B.subtilis, by coupling it with cpYFP in different topological positions and by linking the VSD to the membrane-targeting domain t216. PAPS-Gt was the PAPS variant with the largest fluorescence change upon cell depolarization (-6.3% in the microscope, -13% in the flow cytometer), even if it did not show clear membrane specificity. PAPSGt(D342Y) mutant showed a lower fluorescence change (-5.3% in the flow cytometer), suggesting an important role for D342 in the VSD conformational changes.
Second, the ability of controlling cellular processes with external stimuli is gaining interest with the recent development of optogenetics, magnetogenetics and sonogenetics, but electrical control has lagged behind. An electro-biological interface to complement and enhance bioelectronic systems is proposed with a novel class of proteins named Membrane potential-Activated Targeting Enzymes (MATEs). The first developed MATE is LOTEV, a voltage sensitive TEV protease which cleaves the specific ENLYFQ/S peptide upon externally-induced cell depolarization. The novel protein was obtained by flanking the eukaryotic VSD with the two halves of a split TEV protease (nTEVp and cTEVp). LOTEV showed to increase its proteolytic iv activity by +8.6% in HeLa cells, when the cell membrane was stimulated with a +60 mV electric stimulus.
| Item Type: | Thesis (PhD) | ||||
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| Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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| Library of Congress Subject Headings (LCSH): | Membrane potentials (Electrophysiology), Membrane proteins, Proteolytic enzymes | ||||
| Official Date: | 29 April 2020 | ||||
| Dates: |
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| Institution: | University of Warwick | ||||
| Theses Department: | School of Life Sciences | ||||
| Thesis Type: | PhD | ||||
| Publication Status: | Unpublished | ||||
| Supervisor(s)/Advisor: | Asally, Munehiro ; Dale, Nicholas | ||||
| Sponsors: | Biotechnology and Biological Sciences Research Council (Great Britain) ; Engineering and Physical Sciences Research Council | ||||
| Format of File: | |||||
| Extent: | xxxi, 158 leaves : illustrations (some colour) | ||||
| Language: | eng |
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