Theophylline Riboswitch for Gene Induction and Repression in Mycobacteria
Date of Award
12-2024
Degree Name
Master of Science
Department
Chemistry
First Advisor
Frederick Stull, Ph.D.
Second Advisor
Benjamin Koestler, Ph.D.
Third Advisor
Todd Freestone, Ph.D.
Access Setting
Masters Thesis-Abstract Only
Restricted to Campus until
12-1-2034
Abstract
Development of genetically modified microorganisms for use as microbial cell factories to produce active pharmaceutical agents has proceeded very rapidly in the last decade, for example insulin is produced by genetically modified S. cerevisiae and human growth hormone is produced by E. coli (Sanchez-Garcia, L, 2016). Mycobacteria are used in the steroid industry to produce steroids because of its natural ability to degrade sterols through their metabolic pathways, allowing it to efficiently convert sterols into valuable steroid intermediates which can be further modified chemically to create various steroid drugs. This capability, in conjunction with their cell wall structure, allows it to be a highly efficient ‘cell factory’ for steroid production in the pharmaceutical industry. Therefore, there has been an increased interest in developing synthetic biology tools to regulate gene expression in mycobacterial strains. Riboswitches coupled with reporter genes are of interest because they allow for precise control. In this study, I evaluated the adaptation of a synthetic ligand-inducible riboswitch in M. smegmatis and a proprietary Mycobacteria Pfizer (BBG) strain for co-gene induction and suppression. A genetic circuit was constructed to evaluate if the simultaneous induction and repression of fluorescent reporter genes could be achieved using the riboswitch and the addition of the small ligand Theophylline. EmGFP and paprikaRFP were used as reporter genes, with EmGFP used as the reporter for the inducible riboswitch (Ribo-emGFP) and paprikaRFP used for the repressible riboswitch (intra-paprika). The data for these strains indicated that the co- expression of Ribo-emGFP and intra-paprika showed a detectable induction and suppression of the reporter fluorescent signal when the theophylline ligand was added. Ribo-emGFP displayed a 2-fold increase in fluorescent signal upon addition of 2 mM ligand after 12hr in M. smegmatis and similarly a 2-fold increase in fluorescent signal upon addition of 2 mM ligand after 48hr in BBG. Intra-paprika displayed 0.5-fold reduction in fluorescent signal upon the addition of 0.5 mM ligand after 12hrs in M. smegmatis and 48hr in BBG. More work is necessary to optimize full protein expression in the intra-paprika strain due to the~80% loss of protein expression upon the addition of the aptamer sequence. In this study, we demonstrated the induction and repression of two reported genes, GFP and PaprikaRFP, but other gene of interest (GOI) could be used as substitutes to produce steroid intermediates.
Recommended Citation
O'Neill, Teresa, "Theophylline Riboswitch for Gene Induction and Repression in Mycobacteria" (2024). Masters Theses. 5443.
https://scholarworks.wmich.edu/masters_theses/5443
