Part:BBa_K1900002:Experience

TolC is a barrel-like trimer protein found in many 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 protein 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 strain's 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.

The results are summarized below.

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

Antibiotic efflux confirmed by minimum inhibitory concentration:

Phage infection titer with TLS phage:

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