An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures

Abstract

Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn?PBAD to place the highly regulable, arabinose inducible PBAD promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the PBAD promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.Abbreviations: ECL, enhanced chemiluminescence; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; Tn?PBAD, in vitro transposable element, containing the omega interposon, araC and the araPBAD promoter, flanked by 19 bp inverted repeats recognized by Tn5 transposas