E. coli Tools

KIKO Vectors: Large DNA insertion onto the E. coli genome

KIKO vector with expanded MCS showing 4 operons carrying genes of MEP pathwayThe KIKO vector series is used for rapid, efficient integration of very large DNA sequences onto the E. coli genome at well-characterised non-essential insertion loci. These plasmids are particularly useful for introduction of multiple genes and pathways, for example when reconstructing long metabolic pathways for metabolic engineering applications. Read more here and get the plasmids here.

Knock-in/Knock-out (KIKO) vector. Three operons (OP1a, OP1, OP2-H) carrying genes of the MEP pathway, and a single MEP pathway gene (HDR), have been cloned into the multiple cloning site (MCS) for integration into the E. coli genome by homologous recombination. Click to enlarge images.

kikogfp2

 

Genome of E. coli MG1655. Shows locations of KIKO non-essential target loci arsB, rbsAR and lacZ. Inset shows bright green fluorescence produced by engineered strain carrying green fluorescent protein (gfp), xylanase (xyn) and sucrose utilization (csc) genes. See Figures 1, 2 and 5 (Sabri et al., 2013) here.

Sucrose Utilisation Module

Sucrose is a cheap, abundant carbon source – but most lab and industrial strains of E. coli cannot utilise it. We developed a transferable sucrose utilization approach for non-sucrose-utilizing E. coli strains. Read about it here and get the plasmid here.

transferrable-sucrose-cassette-500

pCSCx plasmid. FRT-CmR-FRT (flippase recognition target, chloramphenical resistance gene) cassette and cscAKB genes were cloned into lacZ gene. The Homologous Recombination Cassette for transferable sucrose utilization was amplified from this plasmid. HA = homologous arm.

Genome Sequence and Genome Scale Reconstruction of E. coli W

Circular map of E. coli W chromosome showing position in bp; forward & reverse ORFs, pseudogenes, pseudoknot; large mobile elements, PLEs, prophages; G+C and A+T content.We sequenced the genome of a sucrose-utilising E. coli strain and developed an improved genome-scale reconstruction of E. coli. Read more and download tools here.

Circular map of E. coli W chromosome. Circles from outside to inside: Outer, position in bp; 2nd, 3rd and 4th (blue), forward ORFs, reverse ORFs and pseudogenes, respectively; 5th (green), pseudoknot; 7th, large mobile elements – phage-like elements (PLEs, green), prophages (red); inner, G+C (purple) and A+T (tan) content. See Fig 1, Archer et al (2011), linked above.