Testing the functionality of SIGEX duo-directional reporter plasmid pSPPH21 using an inducible promoter

Abstract

The bidirectional, promoterless vector pSPPH21 constructed by Abrishamkar et al. features the fluorescence reporter genes, GFP and RFP flanking a NruI restriction enzyme site. Although the presence of the reporter genes in the vector were confirmed via sequencing with a few point mutations in the upstream region of the GFP reporter gene, the functionality of pSPPH21 as a reporter vector has not been elucidated. Thus, in this study, the reporting ability of the pSPPH21 vector will be tested by cloning in a T7-lac inducible promoter system and inducing with an IPTG substrate. A lac operator (lacO) constitutively active mutant was also constructed. The responsiveness of the fluorescent reporting genes in the presence of a metabolic substrate can now be tested alongside the plasmid functionality. However, only recombinant pSPPH21 plasmids with the T7 promoter insert in the orientation specific to the GFP reporter gene were generated. Fluorescence emissions by IPTG induction indicate the downstream GFP reporter gene is responsive to upstream gene inserts. This confirmed that the point mutations in the upstream region of the GFP reporter gene does not affect the reporting ability of pSPPH21. The GFP fluorescent emissions generated through IPTG induction of the ligated T7 promoter provides supporting evidence that the constructed bidirectional vector pSPPH21 is functional in the GFP reporter gene orientation. However, the generated inserts also had levels of fluorescent gene expression without induction, which imply that there is an extremely high affinity of the T7 RNA polymerase to the constructed T7 promoter sequence regardless of induction. No clones generated any RFP expression, thus no conclusions can be made regarding its functionality in pSPPH21. The pT7Mut colonies were revealed to contain no T7Mut promoter insert and thus, no fluorescence activity. Therefore, no conclusions can be made whether the promoter is constitutively activated. Design optimizations of inducible promoters to create more functional clones in order to further validate the GFP and RFP functionality and orientation bias of pSPPH21 is recommended.