Evaluation of a Tet-On promoter for control of gene expression in Variovorax paradoxus
Poster Number
15A
Format
Poster Presentation (Research Day, April 30)
Faculty Mentor Name
Paul Orwin
Faculty Mentor Department
Biological Sciences
Abstract/Artist Statement
Toxin-antitoxin (TA) systems are widespread genetic elements in bacteria thought to contribute to many phenotypes. They function by using differential protein stability to control cell lysis during growth. This project is an effort to evaluate a tightly regulated gene expression system using anhydrotetracycline (aTet) as an inducer to study the role of TA systems in strains of Variovorax paradoxus, an important soil bacterium. These preliminary studies using a GFPuv reporter assay will provide the necessary foundation for designing our future experiments to evaluate these functional elements in phenotypes such as biofilm formation and horizontal gene transfer.
These experiments were conducted to determine if gene expression can be controlled within different strains of Variovorax using the plasmid pBBR2k-GFPuv, which contains a fluorescent reporter under the control of the Tet-On promoter. pBBR2k-GFPuv was purified from E. coli and transformed into the different Variovorax paradoxus strains using electroporation. The presence of the plasmid was verified by agarose gel electrophoresis. The transformed strains were induced with various concentrations of aTet and gene expression was measured at 4 and 24 hours using SDS PAGE and fluorescence microscopy. These results were compared to similar experiments in the original E. coli host strain. The fluorescence microscopy showed a clear induction signal, but the accompanying protein signal in the SDS-PAGE was not as readily detected.
These results will be the basis for further development of this transcriptional control system for use in Variovorax, focused on development of tools to evaluate the functions of TA systems in this important organism.
Location
Information Commons, William Knox Holt Memorial Library and Learning Center
Start Date
30-4-2022 10:00 AM
End Date
30-4-2022 12:00 PM
Evaluation of a Tet-On promoter for control of gene expression in Variovorax paradoxus
Information Commons, William Knox Holt Memorial Library and Learning Center
Toxin-antitoxin (TA) systems are widespread genetic elements in bacteria thought to contribute to many phenotypes. They function by using differential protein stability to control cell lysis during growth. This project is an effort to evaluate a tightly regulated gene expression system using anhydrotetracycline (aTet) as an inducer to study the role of TA systems in strains of Variovorax paradoxus, an important soil bacterium. These preliminary studies using a GFPuv reporter assay will provide the necessary foundation for designing our future experiments to evaluate these functional elements in phenotypes such as biofilm formation and horizontal gene transfer.
These experiments were conducted to determine if gene expression can be controlled within different strains of Variovorax using the plasmid pBBR2k-GFPuv, which contains a fluorescent reporter under the control of the Tet-On promoter. pBBR2k-GFPuv was purified from E. coli and transformed into the different Variovorax paradoxus strains using electroporation. The presence of the plasmid was verified by agarose gel electrophoresis. The transformed strains were induced with various concentrations of aTet and gene expression was measured at 4 and 24 hours using SDS PAGE and fluorescence microscopy. These results were compared to similar experiments in the original E. coli host strain. The fluorescence microscopy showed a clear induction signal, but the accompanying protein signal in the SDS-PAGE was not as readily detected.
These results will be the basis for further development of this transcriptional control system for use in Variovorax, focused on development of tools to evaluate the functions of TA systems in this important organism.