Deciphering the Mechanisms of Silver Resistance in Escherichia Coli
Dr. Misty Thomas
Background: Silver has historically been used as an antimicrobial agent in medical and health settings, but due to its widespread use, the threat of resistance is eminent. The potential mechanisms of silver resistance are not well understood, however, our previous work using experimental evolution have shown that mutations within genes that control major outer porin synthesis (ompR), purine synthesis (purL), RNA polymerase (rpoB) and native copper/silver homeostasis (cusS) may contribute to silver resistance in Escherichia coli. Question: This study focuses on understanding the impact of identified mutations in the histidine kinase cusS in the mechanism of silver resistance by setting up the no-SCAR (Scarless Cas9 Assisted Recombineering) CRISPR genome editing technique in the lab for mutant analysis. Methods: Molecular modeling of three of our detected mutations; L12R, T14P and R15L show that they are located near the N-terminus cytoplasmic tail and the fourth N279H is located near the C-terminus in the dimerization domain. Using the no-SCAR CRISPR genome editing technique we are incorporating these point mutations into the cusS gene in the genome of E. coli. First, we designed primers to create a cusS guide RNA in the pKD plasmid using polymerase chain reaction (PCR) to amplify two portions of the plasmid and circular polymerase extension cloning (CPEC) to join the PCR fragments. The pKD-gRNA plasmid was confirmed to be intact after using gel electrophoresis and subsequently transformed into DH5 cells. The pKD plasmid was then transformed into the competent cells containing the cas9 plasmid. The cells containing the pKD and cas9 plasmid will then be induced to produce Cas9 in order to cut the genome, transformed with ssDNA template with the appropriate mutations in order to induce homologous directed repair and insertion of the mutation. Future Directions: After mutant cell lines are confirmed through sequencing we will then do a phenotypic assessment by performing MIC assays for silver resistance as well as trying to understand the fitness cost of these mutations by performing growth and competition assays with the mutants. Broader Impact: Two-component response systems have been proposed to be a potential target for drug development and therefore this work will help us to better preventively understand the mechanisms of silver resistance before it is widespread in nature and possibly have the opportunity to keep it under control
Riddick, Alexys, "Deciphering the Mechanisms of Silver Resistance in Escherichia Coli" (2018). Undergraduate Research and Creative Inquiry Symposia. 36.