Ectopic integration vectors for generating fluorescent promoter fusions in Bacillus subtilis with minimal dark noise.

Stephanie Trauth,Ilka B Bischofs,Dirk-Jan Scheffers

doi:10.1371/journal.pone.0098360

Abstract

Fluorescent protein promoter reporters are important tools that are widely used for diverse purposes in microbiology, systems biology and synthetic biology and considerable engineering efforts are still geared at improving the sensitivity of the reporter systems. Here we focus on dark noise, i.e. the signal that is generated by the empty vector control. We quantitatively characterize the dark noise of a few common bacterial reporter systems by single cell microscopy. All benchmarked reporter systems generated significant amounts of dark noise that exceed the cellular autofluorescence to different extents. We then reengineered a multicolor set of fluorescent ectopic integration vectors for Bacillus subtilis by introducing a terminator immediately upstream of the promoter insertion site, resulting in an up to 2.7-fold reduction of noise levels. The sensitivity and dynamic range of the new high-performance pXFP_Star reporter system is only limited by cellular autofluorescence. Moreover, based on studies of the rapE promoter of B. subtilis we show that the new pXFP_Star reporter system reliably reports on the weak activity of the rapE promoter whereas the original reporter system fails because of transcriptional interference. Since the pXFP_Star reporter system properly isolates the promoter from spurious transcripts, it is a particularly suitable tool for quantitative characterization of weak promoters in B. subtilis.

Highlights

Fusions between promoters and fluorescent reporter genes have emerged as important study tools that serve many purposes in molecular and applied microbiology, systems biology and synthetic biology
The application spectrum of fluorescent gene reporters ranges from the identification and molecular characterization of cis acting elements, the monitoring of gene expression dynamics in real-time in bacterial bulk populations [1] or individual cells [2], the assessment of population heterogeneity in gene expression, cell phenotype mapping in bacterial micro-colonies and biofilms [3,4] to diverse applications of fluorescent protein promoter fusions in biosensors [5]
We tested all color variants from the pXFPamy family of fluorescent amyE integration vectors in Bacillus subtilis [9], the pGFPbglS vector [9] and the empty plasmid of the GFP-promoter library developed for E. coli [1]

Summary

Introduction

Fusions between promoters and fluorescent reporter genes have emerged as important study tools that serve many purposes in molecular and applied microbiology, systems biology and synthetic biology. By reengineering a multi-color set of fluorescent integration vectors for B. subtilis we obtained an up to 2.7-fold improvement of the noise level with respect to the parent reporters.

Results

Conclusion