Date Thesis Awarded
5-2024
Access Type
Honors Thesis -- Access Restricted On-Campus Only
Degree Name
Bachelors of Science (BS)
Department
Biology
Advisor
Margaret Saha
Committee Members
Sarah Day
Mark Forsyth
GuanNan Wang
Abstract
Currently, bioengineers and synthetic biologists focus their research on only a few well-understood bacterial species, namely, E. coli and B. subtilis, species that thrive only in a few, very specific environments. There is a huge need to develop additional model systems capable of operating beyond the standard laboratory conditions. Fast-growing and ubiquitous in soil, Mycobacterium smegmatis, a non-pathogenic species of the Mycobacterium genus, members include Mycobacterium tuberculosis and Mycobacterium leprae, shows immense promise as a synthetic biology chassis with both clinical and environmental applications. However, more groundwork is needed. This project systematically identified and cloned 19 M. smegmatis promoters and characterized 10 of them. During our promoter selection process, in addition to the Mycobacterial promoters already published in the literature, we extracted 12 novel promoter sequences and its corresponding predicted strength from two transcriptome analyses. After we have obtained our promoter sequences, we constructed a dual-channel fluorescence reporter plasmid for each promoter. Every plasmid has the unique test promoter while having an identical control promoter that acts as an internal control. The control promoter acts as a concurrent reference of the effect of these extrinsic factors on the cells’ overall gene expression pattern. We also used fluorophores-calibrated units and relative strength to maximize reproducibility. We hope this collection of well-characterized promoters, spanning a wide range of strength from weak to strong, will increase the accessibility of more precise and tunable gene expression for constructing complex and reliable systems with M. smegmatis, as it has the potential to serve as a fieldable synbio chassis, providing novel solutions tackling these emergent global crises.
Recommended Citation
Fang, Lin, "Construction of a Mycobacterium smegmatis Promoter Library for Therapeutic and Environmental Applications" (2024). Undergraduate Honors Theses. William & Mary. Paper 2201.
https://scholarworks.wm.edu/honorstheses/2201
