Presentation Title

Evaluating the ShkRS Two Component System in Variovorax paradoxus EPS Utilizing BioID

Faculty Mentor

Paul Orwin

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

75

Session

poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

With the rise of bacterial species possessing antibiotic resistant genes the need for new microbial targets are increasingly important. One potential target for antibiotics are two-component regulatory systems (TCS) which are signal transduction systems ubiquitous in many species of bacteria. We utilized a promiscuous biotin ligase protein derived from Escherichia coli, BirA, which allows for proximity-dependent biotinylation of proteins near a fused bait protein to view potential interaction candidates (BioID). We will apply BioID to investigate protein-protein interactions within Variovorax paradoxus EPS, focused on the ShkRS two component system, which is composed of a sensory histidine kinase (ShkS) and response regulator (ShkR) that were previously shown to control swarming motility. We first created a recombinant broad host range plasmid using the pBBR1MCS2 (kanR) backbone with the birA gene translationally fused to our gene of interest (shkR). This was done by Gibson Assembly, which is based on homologous recombination between overlapping complementary sequences introduced by PCR. The construct also contains a c-Myc epitope that can be used for Western Blot analysis. Wild type V. paradoxus EPS was transformed with the vector backbone, a control birA construct, or the fusion plasmid, by electroporation. The successful transformations were verified by plasmid purification and PCR. Expression of the fusion construct was evaluated by SDS-page and Western Blot analysis for the c-myc tag. Finally, planktonic, biofilm, and swarming cultures were examined using a biotin pulse to identify potential targets, which were visualized using a Streptavidin-HRP conjugate.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Evaluating the ShkRS Two Component System in Variovorax paradoxus EPS Utilizing BioID

75

With the rise of bacterial species possessing antibiotic resistant genes the need for new microbial targets are increasingly important. One potential target for antibiotics are two-component regulatory systems (TCS) which are signal transduction systems ubiquitous in many species of bacteria. We utilized a promiscuous biotin ligase protein derived from Escherichia coli, BirA, which allows for proximity-dependent biotinylation of proteins near a fused bait protein to view potential interaction candidates (BioID). We will apply BioID to investigate protein-protein interactions within Variovorax paradoxus EPS, focused on the ShkRS two component system, which is composed of a sensory histidine kinase (ShkS) and response regulator (ShkR) that were previously shown to control swarming motility. We first created a recombinant broad host range plasmid using the pBBR1MCS2 (kanR) backbone with the birA gene translationally fused to our gene of interest (shkR). This was done by Gibson Assembly, which is based on homologous recombination between overlapping complementary sequences introduced by PCR. The construct also contains a c-Myc epitope that can be used for Western Blot analysis. Wild type V. paradoxus EPS was transformed with the vector backbone, a control birA construct, or the fusion plasmid, by electroporation. The successful transformations were verified by plasmid purification and PCR. Expression of the fusion construct was evaluated by SDS-page and Western Blot analysis for the c-myc tag. Finally, planktonic, biofilm, and swarming cultures were examined using a biotin pulse to identify potential targets, which were visualized using a Streptavidin-HRP conjugate.