Project Summary
As of Spring 2024 2023, we are working to remove nalidixic acid resistance from Escherichia coli using CRISPR-Cas9 delivered via bacteriophage M13.
First, we performed mutagenesis on E. coli strand JM101 by growing and selecting for nalidixic acid resistant colonies. We then amplified the gyrA gene from each colony using PCR and outsourced their sequencing. We also amplified and sequenced the gyrA gene of known nalidixic acid resistant E. coli strain JM109.
Next, we analyzed the sequences to see if any of the strains/colonies we grew carried mutations in codon 83 or 87 of gyrA. Single-nucleotide mutations in these codons are known to lead to nalidixic acid resistance, so we looked for known mutations that we could easily target. We decided to work with JM109 since it had a documneted Asp &rarr Asn mutation in codon 87 of gyrA.
Lastly, we have developed a protocol for inserting Cas9, green flourescent protein, a kanamycin resistance gene, and our custom sgRNA into bacteriophage M13. We intend to construct the modified phage and infect our resistant bacteria with it, at which point we will verify that Cas9 and the sgRNA were expressed from the phage genome and have resensitized the bacteria to nalidixic acid.