Duo-directionality of the substrate-induced expression screening vector pSPPH21 confirmed with a lac-operon screen

Abstract

Within the microbial metabolic diversity lies the potential to utilize catabolic genes for medical, industrial and biotechnological applications. Substrate-induced gene-expression (SIGEX) screening vectors are promoter traps used to screen environmental metagenomic DNA libraries for novel catabolic genes. When a substrate-sensitive promoter is ligated into the plasmid and induced by a substrate, the host cell of the library will express a downstream promoter-less fluorescent reporter. Fluorescence activated cell sorting (FACS) can then be used to isolate positive clones for further study of the ligated promoter and to determine its associated catabolic genes. Despite their importance, traditional SIGEX vectors are limited to detecting promoters ligated in the same 5’ to 3’ direction as the reporter gene. This led to the development of pSPPH21, a duo-directional SIGEX plasmid, by Abrishamkar et al. It was designed to contain two oppositely oriented fluorescent reporters, green fluorescent protein (GFP) and red fluorescent protein (RFP) to allow for the detection of promoters ligated in either direction. We tested the proposed duo-directional functionality of pSPPH21 by using the well-studied inducible promoter of the lac operon. Fluorescence imaging and quantification on a plate reader confirmed that pSPPH21 expresses both GFP and RFP upon induction with the allolactose analog isopropyl-β-D-thiogalactopyranoside (IPTG). We further confirmed that GFP and RFP expression occurs at a 1:1 ratio as expected from the directionally unbiased blunt-end ligation. With the proof-of-concept established, further optimization steps can now be undertaken in preparation for an experimental DNA library screen.