Construction and validation of the Tn5-PLtetO-1-msfGFP transposon as a tool to probe protein expression and localization

Abstract

In this study we report the design, construction and validation of a novel transposon aimed to systematically screen for protein localization and expression patterns in prokaryotes using fluorescence microscopy. Upon random insertion in an open reading frame in the proper frame and orientation, the transposon creates an N-terminal fluorescent protein fusion to the msfGFP reporter. Moreover, in order to examine the localization of fusion proteins whose native expression might be too low or absent, the transposon was fitted with a P_LtetO-1 promoter that makes the expression of the generated fluorescent protein fusions controllable by anhydrotetracycline. Importantly, upon flipping out the P_LtetO-1 promoter and neighboring antibiotic resistance marker, an in-frame "sandwich" msfGFP fusion is created in which the N- and C-terminal portions of the targeted protein are again controlled by its native promoter.

Keywords: Anhydrotetracycline inducible promoter; Cellular localization; Fluorescent protein fusion; Transposon mutagenesis.