Escherichia coli Flagellar Genes as Target Sites for Integration and Expression of Genetic Circuits

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.

Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from Escherichia coli to Bacillus subtilis

“…The genetic circuit Repr-ts-1 consists of the thermosensitive lambda repressor, strong constitutive promoter, RBS and terminator from the Registry of Standard Biological Parts (http://parts.igem.org/Main_Page) [20] (Fig 1). The genetic circuits L1, L2, L3, L12, L13, and L123 encode lysis genes of the bacteriophages MS2, ΦX174, lambda, MS2+ΦX174, MS2+lambda, and MS2+ΦX174+lambda, respectively.…”

“…Oligonucleotide primers were synthesized by Sigma-Aldrich. PCR amplified DNA fragments were assembled in a 5.2 μl final reaction volume using the modified Gibson Assembly procedure [20, 23, 24]. DNA assemblies were confirmed by PCR amplification and sequencing.…”

“…DNA integrations into E . coli chromosome and BACs were performed using the streamlined lambda Red system-mediated method described previously [20, 27, 28]. Briefly, E .…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…First, the DNA fragment is cloned into pSB1K3(FRTK) next to the kanamycin resistance cassette flanked by Flippase recombinase target (FRT) sites and amplified with sequences homologous to the target loci on the E. coli chromosome. [74] Next, the IPTG-inducible lambda Red recombinase system on plasmid pKM208 mediates DNA integration into the E. coli chromosome. Finally, plasmid pCP20-borne Flp recombinase ''flips out'' the kanamycin resistance cassette from the chromosome.…”

“…pSB1K3(FRTK) has been used to integrate synthetic genetic circuits into a number of loci in the E. coli flagellar regions 1, 2, 3a and 3b. [74][75][76] This led to the identification and validation of suitable integration targets in the E. coli flagellar regions, which support high integration efficiency and expression of integrated genetic circuits. [74][75][76] Genome assembly Recent advances in DNA assembly have paved the way for the construction of whole genomes.…”