Development of a Biosafety Level 1 Model for Intimin Expression in Escherichia coli BL21

Abstract

Intimin, an outer membrane protein encoded by the eae gene, plays a critical role in the pathogenesis of attaching and effacing lesions by enteropathogenic Escherichia coli. Although its function in host colonization is well established, the structural characterization of intimin is not. The structure of intimin can be further studied by expressing the eae gene in non-pathogenic E. coli. We aimed to create a plasmid with an inducible promoter to facilitate controlled expression of intimin in a non-pathogenic host, BL21 E. coli. The eae gene encoding intimin was successfully cloned into pBAD24, downstream of an arabinose-inducible promoter and upstream of a 6xHis tag to create the pCCAN-eae plasmid. pCCAN-eae expression was induced in BL21 E. coli, and the death of cells was measured as a substitute for intimin expression. Higher levels of arabinose and therefore intimin expression were found to induce cell death.  This biosafe, inducible system provides a tool for future investigations into intimin’s structure, and role in bacterial adhesion, and host-pathogen interactions.