Preliminary Steps Toward Establishing a Two-Plasmid System to Drive Lactobacillus plantarum Promoter Expression in Escherichia coli Using Lactobacillus plantarum Sigma Factor, RpoD

Abstract

The use of bacterial genes in biotechnology is widespread, but the precise functions of many genes belonging to environmental microbes remain unknown in function, as methods to culture many bacteria are yet to be optimized. To induce the expression of heterologous genes from unculturable bacteria, Gaida et al. introduced a two plasmid system to drive heterologous promoter expression using the Lactobacillus plantarum sigma factor, RpoD, in E. coli (Gaida et al., Nature Communications, 2015). This successfully resulted in increased expression of the Lactobacillus plantarum metagenomic libraries inserted into Escherichia coli. Given the promise of this technique, we aimed to set up this two plasmid system in our laboratory. In this paper, we describe key features of the two plasmid system and progress with respect to transforming and propagating the plasmids in Escherichia coli host strains. The two plasmid system consists of a Lactobacillus plantarum sigma factor expression vector, pLplσ, and a second plasmid responsible for carrying heterologous promoters while also providing a method to quantify heterologous promoter expression via a promoter-gfp trap concept. For the latter plasmid, prior to the insertion of heterologous promoters, pLR-GFP serves as the promoterless-GFP trap destination vector. The pUC-LR-GFP destination vector is near identical in construction as pLR-GFP, but differs only in the presence of a lac promoter located upstream of the segment where heterologous promoters are later inserted, such that the heterologous promoter-gfp trap segment are under its control, and when induced will result in transcription and GFP expression. pUC-LR-GFP thus acts as a positive control and validates the proposed concept. pControl is the negative control for pLplσ, with near identical construction as pLPLσ, but lacking rpoD. We were able to successfully propagate  pControl and pLPLσ in E. coli DH5ɑ. However, due to the presence of the toxic ccdB gene in pLR-GFP, it was instead propagated in the ccdB resistant E. coli JM109 strain. We also performed restriction digest analysis of each plasmid, and confirmed the identity of pLPLσ via Sanger sequencing. Future steps to fully establish this two plasmid system in the laboratory include utilizing the Invitrogen Gateway technique to clone heterologous promoters into pLR-GFP, co-transformation of pLPLσ and pLR-GFP into Escherichia coli DH5ɑ, and using flow cytometry to quantify and analyze GFP expression in the presence or absence of RpoD.