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 proteins' ability to function in E. coli.

Characterization of tolC parts by WLC-Milwaukee 2018

WLC-iGEM Continued working with E. coli tolC to better characterize the function of this part and analyze it's antibiotic efflux capabilities In previous characterizations of this part, we have shown that TolC proteins from other gram-negative bacteria can be expressed in E. coli and act differently with regard to the ability to efflux antibiotics. We showed previously that the TolC protein from Klebsiella pneumoniae has increased sensitivity to antibiotics including Novobiocin (see Figure 1 below). This is interesting since the Klebsiella TolC protein has a relatively high similarity and identity to the E. coli TolC protein (Table 1). We used bioinformatics approaches to identify eight amino acids in the Klebsiella TolC protein that we could change to potentially reduce its sensitivity to antibiotics so that it is more similar to that of E. coli TolC. We performed site-directed mutagenesis on the Klebsiella tolC clone and performed disk sensitivity assays again (Figure 2) which showed that the mutations increased the resistance to antibiotics. To further characterize part BBa_K1900002, we performed a reciprocal experiment where we mutated the E. coli tolC gene to put the Klebsiella TolC residues into TolC at the same specific locations. We show in Figure 3 that changing these specific amino acids in the E. coli TolC protein do not significantly alter protein levels compared to wild-type E. coli TolC. We then performed disk sensitivity assays (Figure 4) to determine if these amino acid changes increased sensitivity to antibiotics similar to the Klebsiella TolC protein. The results show that there is not a significant change in the function of E. coli when the reciprocal mutations are added. This suggests that E. coli TolC function is more able to adapt to these changes than the Klebsiella TolC that likely has other different amino acids that contribute to the phenotypes.

Figure 1

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Table 1

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Figure 2

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