Project Summary
As of Spring 2025, we are working to remove nalidixic acid resistance from Escherichia coli using CRISPR-Cas9 delivered via bacteriophage M13. We are also analyzing unusual gyrA sequencing results after sequencing spontaneously resistant E. coli strain JM101 cells cultured in a nalidixic acid minimum inhibitory concentration (MIC) assay.
First, we performed mutagenesis on E. coli strain JM101 by growing and selecting for nalidixic acid resistant colonies in a naladixic acid MIC assay. The same MIC assay was used to confirm the resistance of known nalidixic acid resistant E. coli strain JM109. We then amplified the beginning of the coding sequence of the gyrA gene from each spontaneously mutant JM101 colony using PCR and outsourced their sequencing. We also amplified and sequenced the gyrA gene of strain JM109.
Next, we analyzed the sequences to see if any of the 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, are common in wild and pathogenic populations, and are easily targetable with CRISPR-Cas9 gene editing. We decided to work with strain JM109 since it had a documneted Asp → Asn mutation in codon 87 of gyrA, while all of the mutant JM101 colonies had mutations which were found to be rare by previous studies.
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.