Multiple deletions arise from attempted insertion of the salicylate nahR/Psal biosensor into a duo-directional SIGEX vector

Abstract

To address the ongoing problem of plastic accumulation in marine environments, there is a need to identify microbial communities capable of degrading plastic associated pollutants and characterize the inducible systems that they use for applications in synthetic biology. Substrate-induced gene expression  reporters have been used as a screening tool to find inducible promoters sensitive to plastic-associated compounds. A duo-directional substrate-induced gene expression reporter (pSPPH21), with fluorescent reporter proteins on either side of the insertion site, is theoretically capable of searching for both uni- and bi-directional promoters and would function independent of the insert orientation. This study evaluates the functionality of pSPPH21 as a tool to screen environmental samples for promoters neighboring plastic-degrading enzymes. To this end, the nahR/Psal bi-directional promoter system was inserted into pSPPH21 (pSPPH21::nahR/Psal) and subsequently transformed into competent Escherichia coli cells. Colonies were induced with salicylate, a breakdown product of naphthalene, and measured for fluorescence activity. Colonies with the pSPPH21::nahR/Psal construct generally responded to salicylate induction in a concentration dependent manner. A response was not observed by salicylate alone or upon induction of the empty pSPPH21 vector. In practice, the nahR/Psal biosensor did not respond to water stored in a polyethylene terephthalate container for 6 days. Despite the observed responses, sequencing results of the tested colonies displayed no insertion of the nahR/Psal fragment at the NruI site and multiple deletions within or surrounding the NruI site.