Functional characterization of IPTG and salicylate inducible systems promotes utility of SIGEX duo-directional reporter vectors in metagenomic screening

Abstract

Precisely controlling and tuning inducible promoter responses to environmental stimuli is an important step in the engineering of synthetic gene circuits and biological systems for metagenomic applications. A duo-directional SIGEX plasmid using GFP and RFP reporters has been designed to address the unidirectional nature of biosensors. Studies have provided proof-of-concepts for IPTG and salicylate inducible pSPPH21-modified biosensors (pSPPH21-lacI and pSPPH21::nahR/Psal) by detection of GFP and RFP. Our incomplete understanding of SIGEX’s sensitivity to different concentrations of inducers remains unknown. In this study, we explore the reporter expression dynamics of two SIGEX vectors controlled by different inducible promoters to investigate relationships between concentration and the effect on downstream gene reporter expression. We found that pSPPH21-lacI is responsive and hence sensitive across a vast range of IPTG concentrations ranging from 10 µM to 1 mM, with greatest inducible promoter responses seen between 8-10 hours. We also found that the pSPPH21::nahR/Psal plasmid is non-functional and is unable to be induced by salicylate. In sum, our work shows that SIGEX is sensitive across a wide range of inducible substrate concentrations. With a functional characterization of SIGEX established, further optimization steps can now be undertaken in preparation for high-throughput DNA promoter screening from an experimental metagenomic sample.