Title

Evaluation of a Tet-On promoter for control of gene expression in Variovorax paradoxus

Poster Number

15A

Lead Author Major

Pre-Dentistry

Lead Author Status

Sophomore

Second Author Major

Biological Sciences

Second Author Status

Junior

Third Author Major

Biological Sciences

Third Author Status

Junior

Fourth Author Major

Biological Sciences

Fourth Author Status

Junior

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

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Apr 30th, 10:00 AM Apr 30th, 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.