Part:BBa_K1900002:Experience

TolC is a barrel-like trimer protein that spans the outer membrane in E. coli, as well as many other gram-negative bacteria. This protein is of particular interest because of its role in antibiotic resistance. Specifically, TolC is known form an efflux pump when integrated with AcrA and AcrB. Another interesting function of the protein is bacteriophage infection by the TLS phage. We wanted to test the E. coli TolC functions against other gram-negative bacteria's TolC proteins. To improve on the characterization of the E. coli tolC part, our 2017 iGEM team analyzed functions of the E. coli TolC protein in comparison with other species' TolC proteins expressed in a strain of E. coli lacking the gene. Three assays were done with various antibiotics: minimum inhibitory concentration, zones of inhibition, and TLS phage infection. Key findings include that certain strains' TolC proteins do not function well in E. coli, while others do. This leads to indications surrounding which regions of TolC are necessary for antibiotic efflux, and which may be necessary for bacteriophage infection. With some bioinformatic analysis of the variance in amino acid sequences of the TolC proteins, more precise conclusions can be drawn regarding which exact sites integrate with AcrA and AcrB, as well as the mechanism for bacteriophage infection in the extracellular loops. The results are summarized below.

Antibiotic efflux demonstrated by zones of inhibition with Kirby-Bauer assays:

Antibiotic efflux capabilities confirmed by minimum inhibitory concentration:

Phage infection titer with TLS phage:

The amino acid sequences for regions necessary for AcrA interaction (top) and TLS phage infection (bottom) are shown. Conservation in these sequences translate to the strains' TolC's ability to function in E. coli.

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