Date of Award
2024
Degree Type
Thesis
Degree Name
Master of Science in Biological and Environmental Sciences (MSBES)
Specialization
Cell and Molecular Biology
Department
Cell & Molecular Biology
First Advisor
Kathryn Ramsey
Abstract
Francisella tularensis is a Gram-negative, facultative, intracellular bacterium which causes the disease tularemia. Previous research demonstrated that F. tularensis ribosomes are heterogeneous and that a specific ribosomal protein, bS21-2, regulates translation by interacting with the leader sequences of mRNAs (Trautmann and Ramsey 2022). Studies using reporter assays in bacterial cells (in vivo) made significant progress towards identifying leader sequences that confer regulation by bS21 (Trautmann et al. 2023), but we would like to recapitulate these results using purified ribosomes (in vitro) and develop a more comprehensive model of regulation by bS21.
The goal of my research was to develop an in vitro assay for translation using F. tularensis Live Vaccine Strain (LVS) ribosomes with the NEB PURExpress ® in vitro translation kit. To achieve this goal, I developed a sensitive and easily modifiable reporter construct, purified active ribosomes from Escherichia coli and F. tularensis LVS, and optimized the reproducibility of the in vitro assay.
For my research project, I used standard cloning techniques to generate a DNA template for the in vitro assay that encodes a luminescent reporter and is sensitive and easy to modify. I also purified salt-washed E. coli ribosomes and determined that they were translationally active in the assay. Then, I replicated these results using ribosomes from multiple purification attempts, validating that we can reproducibly isolate active ribosomes. In addition, I demonstrated that E. coli ribosomes are inhibited by ribosome-targeting antibiotics, indicating that we can use the assay to measure translation inhibition. Next, I purified ribosomes from F. tularensis and made enhancements to increase ribosome concentration and yield. Then, I found that F. tularensis ribosomes appeared to aggregate more readily than E. coli ribosomes and that this aggregation seemed to impact translational activity. After further testing, I modified the amount of F. tularensis LVS ribosomes used in the in vitro assay to reduce aggregation and obtain a significant and reproducible signal.
The optimized in vitro assay that I developed will be used in future research to measure translation efficiency of mRNA leader sequences by F. tularensis ribosomes with specific homologs of bS21 and will contribute to a better understanding of how bS21 homologs regulate translation in F. tularensis.
Recommended Citation
Moore, Benjamin, "EVALUATING FRANCISELLA TULARENSIS TRANSLATION IN VITRO" (2024). Open Access Master's Theses. Paper 2573.
https://digitalcommons.uri.edu/theses/2573
