Identification of mutations in the bacterium Pseudomonas aerugionosa that confer resistance to a novel antimicrobial peptide DASamp2 via inverse PCR
Advisor Information
Donald Rowen
Location
Dr. C.C. and Mabel L. Criss Library
Presentation Type
Poster
Start Date
4-3-2016 10:45 AM
End Date
4-3-2016 12:15 PM
Abstract
With the widespread use of antibiotics to treat infections, previously sensitive pathogenic bacteria were observed to evolve resistance to the antibiotics being used. Extremely resistant bacteria have become increasingly prevalent in recent years, and have prompted a scramble to discover new antimicrobial drugs. The antimicrobial peptide DASamp2 was identified as a promising new antibacterial drug that was effective against Pseudomonas aeruginosa and other bacteria. We are seeking to try to identify its target or mechanism of action. Dr. Rowen’s lab isolated 10 mutants of the bacteria that showed increased resistance to the peptide. The cells were mutated by insertion of a mini-transposon. Identification of the genes mutated, which induced resistance, should reveal clues as to the target of DASamp2. I am seeking to identify the gene mutated in two of the isolated mutants, H30 and H36. To determine the gene mutated, I have been trying inverse PCR to amplify the genomic sequences flanking the transposon. For this procedure DNA was isolated from mutants with Wizard Genomic DNA Purification kit and cut with restriction enzymes at sequences selected matching the transposon. This genomic DNA was then circularized using T4 DNA ligase, then inverse PCR technique amplified DNA. Purifying the PCR product and sequencing can determine the nucleotide sequence of adjacent DNA, the gene of interest.
Identification of mutations in the bacterium Pseudomonas aerugionosa that confer resistance to a novel antimicrobial peptide DASamp2 via inverse PCR
Dr. C.C. and Mabel L. Criss Library
With the widespread use of antibiotics to treat infections, previously sensitive pathogenic bacteria were observed to evolve resistance to the antibiotics being used. Extremely resistant bacteria have become increasingly prevalent in recent years, and have prompted a scramble to discover new antimicrobial drugs. The antimicrobial peptide DASamp2 was identified as a promising new antibacterial drug that was effective against Pseudomonas aeruginosa and other bacteria. We are seeking to try to identify its target or mechanism of action. Dr. Rowen’s lab isolated 10 mutants of the bacteria that showed increased resistance to the peptide. The cells were mutated by insertion of a mini-transposon. Identification of the genes mutated, which induced resistance, should reveal clues as to the target of DASamp2. I am seeking to identify the gene mutated in two of the isolated mutants, H30 and H36. To determine the gene mutated, I have been trying inverse PCR to amplify the genomic sequences flanking the transposon. For this procedure DNA was isolated from mutants with Wizard Genomic DNA Purification kit and cut with restriction enzymes at sequences selected matching the transposon. This genomic DNA was then circularized using T4 DNA ligase, then inverse PCR technique amplified DNA. Purifying the PCR product and sequencing can determine the nucleotide sequence of adjacent DNA, the gene of interest.
Additional Information (Optional)
Winner of Meritorious Undergraduate Poster Presentation