Developing a BrkA-based Protein Expression System in Escherichia coli Using the Mini-Tn7 Vector System

Abstract

Autotransporters are a family of proteins secreted to the cell surface of Gram-negative bacteria that require only the autotransporter protein itself for transport. Many of these autotransporters, such as Bordetella resistance to killing (BrkA) protein found in the organism Bordetella pertussis, have applications in the realm of protein expression and presentation. Use of autotransporters like BrkA in protein expression systems has largely been confined to the context of plasmid-based systems, which have known limitations including harming bacterial host cell fitness. This has sparked interest in alternatives that bypass these limitations, such as using transposons to mediate insertion of desired genes into the bacterial genome. We leveraged a mini-Tn7 system to introduce a single copy of brkA into the Escherichia coli genome. 6xHis tagged-brkA under the control of the lac promoter was amplified and inserted into a linearized mini-Tn7 vector via Gibson assembly. DH10B E. coli cells were transformed with our Gibson product together with a helper plasmid to catalyze transposition, with transposition being demonstrated by the presence of gentamicin resistance colonies. lac/brkA-6xHis was inserted into our transposon vector, and cells transformed with the vector demonstrated gentamicin resistance; however, we were unable to confirm the single insertion of brkA into the vector and its specific incorporation into the E. coli genome at the expected site through PCR. Sequencing results for our Gibson assembly product also warrant further investigation.