A library of reporters of the global regulators of gene expression in Escherichia coli

Abstract

ABSTRACT The topology of the transcription factor network (TFN) of Escherichia coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for green fluorescent protein (GFP) fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli. Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.IMPORTANCECells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries.

Keywords

• global regulators of gene expression
• transcriptional reporters
• synthetic single-copy plasmids
• native promoters
• fluorescent proteins
• strain library