Construction of SIGEX duo-directional reporter plasmid pSPPH21

Abstract

Substrate-induced gene expression (SIGEX) methods use gene reporter systems to screen metagenomic libraries and identify novel catabolic genes. Commonly, these reporter systems incorporate promoter-less fluorescent protein genes downstream of an overhang-producing restriction site which allows metagenomic DNA fragments to be inserted and analyzed. SIGEX methods, however, lack the ability to detect genes which are inserted in the reverse orientation based on the unidirectional design of the SIGEX vector as determined by the overhangs produced during the cloning process. The insertion of a second oppositely oriented fluorescent reporter gene and a blunt-end producing restriction site would circumvent this limitation. In this study, we adapted a vector design from UBC iGEM to construct a duo-directional SIGEX plasmid reporter system (pSPPH21) containing green fluorescent protein (GFP) and red fluorescent protein (RFP) reporter genes. To build plasmid pSPPH21, a 766 base pair DNA fragment containing an NruI restriction site and an RFP gene was synthesized, digested, and cloned into the pSB1C3 vector containing a GFP and chloramphenicol resistance gene to construct the duo-directional reporter vector. Successful assembly of the vector was verified using gel electrophoresis and Sanger sequencing. The location of the NruI restriction site between the oppositely oriented GFP and RFP genes allows for high efficacy cloning of inducible promoters from metagenomic libraries and for the identification of novel catabolic genes.