Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

Mario Juhas,Gillian M Fraser,Lewis D B Evans,Orr Yarkoni,Joe Frost,Peter W Davenport,James W Ajioka,Mark Isalan

doi:10.1371/journal.pone.0111451

Abstract

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

Highlights

The Gram-negative rod-shaped bacterium Escherichia coli K12 is one of the most common microbes used for bioproduct manufacturing, metabolic engineering and as a chassis for Synthetic Biology devices [1,2,3,4,5]
We selected the flagellar regions as putative integration sites based on RNA-polymerase profiling (Figure 1 and Figure S1) and the observation that they are located in highly expressed genomic regions that are enriched for interactions with DNA-binding proteins [9]
Repr-ts-1 was modified with flanking sequences derived from four genes in the flagellar region to produce a series of constructs ready for integration into the E. coli genome

Summary

Introduction

The Gram-negative rod-shaped bacterium Escherichia coli K12 is one of the most common microbes used for bioproduct manufacturing, metabolic engineering and as a chassis for Synthetic Biology devices [1,2,3,4,5]. To test the suitability of chromosomal loci for expression of genetic constructs, we chose to integrate a simple genetic construct consisting of the gene encoding the thermosensitive l repressor cI857 and a constitutively active upstream promoter and ribosome binding site (RBS) elements from the Registry of Standard Biological Parts (Figure S2). At 30uC the repressor protein negatively regulates gene expression from the bacteriophage l pR and pL promoters, but raising the temperature to 42 uC relieves repression allowing transcription from pR and pL [11]. We integrated this small genetic device into four promising sub-loci within flagellar region 3a. We propose fliT as standard site for the integration and characterisation of genetic devices

Methods

Results

Conclusion