Cloning of TA system loci from Variovorax paradoxus EPS and evaluation of toxicity assays for gene function analysis.
Poster Number
5A
Format
Poster Presentation
Faculty Mentor Name
Paul Orwin
Faculty Mentor Department
Biological Sciences
Graduate Student Mentor Name
Annie Lin
Graduate Student Mentor Department
Biological Sciences
Abstract/Artist Statement
Toxin-antitoxin (TA) systems are widespread elements in bacteria genomes that function in bacterial physiology, stress response, and defense against antimicrobial substances. They are composed of an inhibitory toxin that affects essential cellular processes and a neutralizing antitoxin. The toxin is more stable than the antitoxin protein, meaning that changes in expression level can lead to cell lysis. We hypothesize that these systems play a role in biofilm development.
Our goal was to clone predicted toxin-antitoxin genes from Variovorax paradoxus EPS into an inducible genetic system using arabinose and glucose to control expression. The aim is to develop a system for testing whether predicted chromosomal TA elements are actual TA systems, and how these systems affect bacteria physiology.
We selected eight predicted TA system elements from a database housing the V. paradoxus EPS genome, along with a control TA element (pemIK). We designed PCR primers to amplify each region based on a prediction of the TA system structure. Gibson assembly was conducted to clone predicted TA sequences from V paradoxus EPS into the pBBR-8k vector, with the end goal of expressing the gene in an inducing cell. The plasmid was then sequenced to confirm that the TA system sequence was correctly implemented in the plasmid.
Seven of the eight targeted cloning experiments were successful. Slow growing colonies from the 3227-28 target did not contain plasmid. An annotation error in the predicted TA sequence resulting in the cloning of a toxic element. Two different assays were performed on the control plasmids to validate TA system screening approaches.
Location
Information Commons, William Knox Holt Memorial Library and Learning Center
Start Date
29-4-2023 10:00 AM
End Date
29-4-2023 1:00 PM
Cloning of TA system loci from Variovorax paradoxus EPS and evaluation of toxicity assays for gene function analysis.
Information Commons, William Knox Holt Memorial Library and Learning Center
Toxin-antitoxin (TA) systems are widespread elements in bacteria genomes that function in bacterial physiology, stress response, and defense against antimicrobial substances. They are composed of an inhibitory toxin that affects essential cellular processes and a neutralizing antitoxin. The toxin is more stable than the antitoxin protein, meaning that changes in expression level can lead to cell lysis. We hypothesize that these systems play a role in biofilm development.
Our goal was to clone predicted toxin-antitoxin genes from Variovorax paradoxus EPS into an inducible genetic system using arabinose and glucose to control expression. The aim is to develop a system for testing whether predicted chromosomal TA elements are actual TA systems, and how these systems affect bacteria physiology.
We selected eight predicted TA system elements from a database housing the V. paradoxus EPS genome, along with a control TA element (pemIK). We designed PCR primers to amplify each region based on a prediction of the TA system structure. Gibson assembly was conducted to clone predicted TA sequences from V paradoxus EPS into the pBBR-8k vector, with the end goal of expressing the gene in an inducing cell. The plasmid was then sequenced to confirm that the TA system sequence was correctly implemented in the plasmid.
Seven of the eight targeted cloning experiments were successful. Slow growing colonies from the 3227-28 target did not contain plasmid. An annotation error in the predicted TA sequence resulting in the cloning of a toxic element. Two different assays were performed on the control plasmids to validate TA system screening approaches.