In Vivo Recombineering for Insertion of Chromosomal Mutations in Two-Component Response Systems

Faculty Mentor

Dr, Joseph Graves Jr. and Dr. Misty Thomas

Document Type


Publication Date

Spring 2023


Two-component response systems (TCRS) are important in allowing bacteria, such as E. coli, to sense physical and chemical changes. This process is facilitated by histidine kinases, which elicit the correct signal in response to environmental changes. Current literature demonstrates that TCRS play a role in bacterial acclimation, however very little is known about their role in adaptation. During long term exposure to silver nitrate, a potent antimicrobial, multiple papers have identified adaptive mutations in EnvZ/OmpR and CusS/CusR, therefore this project focuses on using the EnvZ/OmpR TCRS to better understand TCRS role in adaptation. We hypothesize that mutations in two-component response systems will cause the organism to become less fit as they acquired their new phenotype which is resistance to silver. The goal of this project is to insert chromosomal mutations into E. coli K12 to study the impact that these mutations have on fitness of the organism. Using in vivo recombineering technology, we inserted chromosomal mutations into E. coli K12. In the most recent recombineering run, we used the original MG1655 strain of E. coli K12 as the WT and attempted to insert the del196 OmpR mutation. To identify if the mutations were inserted properly, we performed colony polymerase chain reaction (PCR), and E.Z.N.A bacterial DNA kit to isolate the chromosome, and PCR to amplify the gene. These PCR results were then verified by gel electrophoresis. Unfortunately, after sending off our PCR product from the bacterial DNA kit the sequencing results showed that we were not successful in inserting the del196 mutation for OmpR. However, next steps include trying to insert the mutation through transformation of the mutants on a plasmid. After being successful in inserting the mutation, the goal is to study how these mutations affect the fitness of the organism by measuring growth rate, competitive behavior, and reversion rate.


Lauren Thomas, Co-First Author
Destinee Harris, Co-First Author

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