DOI
https://doi.org/10.25772/X56V-DY32
Defense Date
2022
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Ashton Cropp
Abstract
1. Developing transcriptional repressor protein, MphR based macrolide antibiotic biosensors as high throughput screening methods
Macrolides are a class of antibiotics which are in high demand for clinical applications. They are biosynthesized via large assembly line Polyketide Synthases (PKS) arranged in a modular fashion. There is an urgent need for novel macrolides due to the growing resistance of bacteria towards antibiotics. Combinatorial biosynthesis is an approach to reprogram the PKS modules to diversify macrolides. However due to the size and the complexity of PKSs combinatorial biosynthesis approach is challenging. Directed evolution is an alternative approach where large libraries of enzyme variants are designed and screened. It is important to develop high throughput screening methods to screen such enzyme libraries. MphR is a transcriptional regulator protein 14 which represses the transcription upon binding to a DNA promoter sequence. It can bind to macrolides promiscuously and release from the DNA promoter de-repressing the transcription. In this research a gene expressing the Green Fluorescence Protein (GFP) is incorporated to the biosensor system. Macrolides will bind to MphR, that will de-repress a gene cassette with gfp gene leading to green fluorescence in the presence of macrolides. This study focuses on improving the sensitivity of the MphR biosensor using a gene knockout approach. Furthermore, it focuses on developing a ratio metric biosensor for the simultaneous detection of erythromycin and clarithromycin which is a semi-synthetic derivative of erythromycin and discusses application of the biosensor. Based on the promiscuity of MphR this provides a promising strategy to engineer biosensors for the detection of macrolides.
2. Recombinant protein expression with acylated lysine post translational modifications to expand the genetic code
Post Translational Modifications have a massive role in expanding the proteome. It is important to study the post translational modifications in depth in order to understand the functionality and activity of the recombinant proteins. This study focuses on lysine acylation post translational modifications. β-hydroxy butrylation post translational modification on lysines were previously identified in histone proteins. This modification shows direct coupling of metabolism to gene expression. To study these modifications in-depth, it is critical to obtain homogeneously modified proteins. This study presents a method to incorporate acylated lysines to recombinant proteins. In this study acylated lysines 3-hydroxy butryl lysine, isobutryl lysine and isovaleryl lysine were successfully incorporated into super folder GFP and histone h3 proteins using Pyrlysine tRNA. tRNA synthetase orthogonal pair
Rights
Jayani A. Christopher
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-12-2022
Comments
The thesis is written as two major sections describing two major projects, developing biosensors for macrolide antibiotics and genetic code expansion with unnatural amino acids.