Construction of luxCDABE reporter plasmids to investigate regulation of the acrAB and acrEF operons by the AcrS repressor in Escherichia coli BW25113


Zoe O’Neill, Olivia Bulka, Megan Marziali, Ryan Nah​

Volume 4
Fall 2017 / Winter 2018

SUMMARY The acrAB and acrEF operons of Escherichia coli encode multidrug efflux pumps capable of efficiently exporting a wide variety of antibiotics. The repression of acrAB and acrEF by regulator, AcrS, has not yet been completely elucidated. In order to investigate the role of AcrS in the regulation of the acrAB and acrEF operons, reporter plasmids containing the promoter sequences of these operons were constructed. Promoter regions of acrAB and acrEF were first amplified and stored in the pCR™2.1-TOPO® vector. Sequencing confirmed that the complete promoter regions of both acrAB and acrEF were successfully cloned. In order to assess regulation of this system in future experiments, the PCR-amplified promoters were cloned into pCS26. The pCS26 vector contains a promoter-less reporter luxCDABE operon. Here, we report that we successfully cloned the acrAB and acrEF operon promoter upstream of the luxCDABE operon in pCS26 using Gibson assembly. These constructs can be used in future experiments monitoring change in acrAB and acrEF gene expression in response to AcrS.