The Library
Heterologous repressors for controlled expression of recombinant proteins in Escherichia coli
Tools
Taylor, Daniel (2022) Heterologous repressors for controlled expression of recombinant proteins in Escherichia coli. PhD thesis, University of Warwick.
|
PDF
WRAP_Theses_Taylor_2022.pdf - Submitted Version - Requires a PDF viewer. Download (8Mb) | Preview |
Official URL: http://webcat.warwick.ac.uk/record=b3944069
Abstract
MmfR is a transcriptional repressor protein from Streptomyces coelicolor which regulates the production of the antibiotic methylenomycin. MmfR is chemically induced by methylenomycin furans (MMFs), a family of stable, low-molecular-weight diffusible molecules. MmfR has previously been proven to function when produced in Escherichia coli.
In the work described in this thesis, the use of MmfR as a transcriptional regulator for recombinant protein production in E. coli was explored, in tandem with its binding site and inducer, as an alternative to the traditional LacI/LacO/IPTG inducible expression system. It was necessary to establish whether MmfR could be expressed constitutively by E. coli, and whether it could be as effective a repressor as LacI for biotechnological applications.
A recombinant protein production construct was designed de novo to use MmfR as a transcriptional repressor, and to express GFP as a reporter. The construct was assembled, transformed into E. coli, and observed using time-resolved fluorescence. The construct was directly compared to a parallel construct that instead used LacI as its transcriptional repressor.
Experimental data showed E. coli to be capable of growing effectively whilst constitutively expressing MmfR, and MmfR to be able to repress transcription of the reporter gene when the latter was regulated by MmfR’s MARE target sequence. However, MmfR was also observed to be leakier than LacI, less able to repress GFP expression when not exposed to its MMF inducer.
Computational modelling of the expression system in COPASI identified several approaches that could be taken to reduce the expression system’s overall metabolic burden on the host cell. The MmfR expression system shows promise as an alternative to LacI-based expression, but further work is required to establish it as a viable alternative for large-scale recombinant protein production.
Item Type: | Thesis (PhD) | ||||
---|---|---|---|---|---|
Subjects: | Q Science > QH Natural history > QH426 Genetics Q Science > QR Microbiology |
||||
Library of Congress Subject Headings (LCSH): | Transcription factors, Repressors, Genetic, Genetic transcription -- Regulation, Recombinant proteins, Escherichia coli -- Genetics | ||||
Official Date: | August 2022 | ||||
Dates: |
|
||||
Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Corre, Christophe ; Kalvala, Sara | ||||
Sponsors: | Engineering and Physical Sciences Research Council ; Biotechnology and Biological Sciences Research Council (Great Britain) | ||||
Format of File: | |||||
Extent: | 129 pages : colour illustrations | ||||
Language: | eng |
Request changes or add full text files to a record
Repository staff actions (login required)
View Item |