Cloning optimization for substrate-induced gene expression technology

Abstract

Substrate-induced gene expression (SIGEX) is a genetic technique utilized for the isolation of novel substrate-inducible catabolic genes from environmental metagenomic samples. Previous SIGEX plasmids have relied on a single reporter gene for high-throughput detection of novel genes in metagenomic libraries. This limited its use for genetic inserts in the opposite direction of the reporter gene which theoretically occurs in half of all inserts. A recent study by Abrishamkar et al. described the creation of a new duo-directional SIGEX-based plasmid named pSPPH21 by including two fluorescent reporter genes in opposite orientations. This newly created plasmid has yet to be validated in a SIGEX-based experiment. Here we aimed to test the functionality of this plasmid using an inserted inducible lac operon promoter sequence. We were unable to clone the insert however, several strategies were researched and utilized for cloning. We have summarized these strategies and we hope this will serve as a resource for optimizing future implementation of SIGEX technology.