Chromosomal Integration of Heterologous DNA in Escherichia coli with Precise Removal of Markers and Replicons Used during Construction

Abstract

A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain twoFRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomaladhE gene in strains of E. coli K-12 andE. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.

Metabolic responses to recombinant bioprocesses in Escherichia coli

“…(Gregory and Turner, 1993;Turner et al, 1994a) manipulations are easier and less time consuming compared to methodologies where genes are integrated into the chromosome. This may explain why only few methodologies for the insertion of heterologous genes into the host chromosome have been developed (Chiang et al, 2008;Martinez-Morales et al, 1999;Wei et al, 2010;Blaas et al, 2009;Chen et al, 2008). Besides those strategies concerned with the engineering of expression vectors to reduce the metabolic burden in the host cells, together with the increase of protein expression levels, the redesign of coding-sequences of the target products has also been considered.…”

Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C

“…2) was constructed with a removable cat-sacB cassette and the option to include an 18-bp segment of synthetic DNA with stop codons in all reading frames. This plasmid is composed of synthetic sequences and parts of plasmids pLOI2228 (Martinez-Morales et al, 1999), pLOI2511 (Underwood et al, 2002), and pEL04 (Lee et al, 2001;Thomason et al, 2005). Using pEL04 as a template, inside-out PCR was performed with the JMpEL04F1/R1 primers to eliminate unwanted SmaI and BamHI sites between the cat and sacB genes.…”

Replicon‐free and markerless methods for genomic insertion of DNAs in phage attachment sites and controlled expression of chromosomal genes in Escherichia coli

“…For incorporation of heterologous genes into E. coli chromosome, the flippase-based method has been proposed (Martinez-Morales et al, 1999b). This approach relies on the construction of two flanking FRT sites in the suicidal plasmid which carries the guide DNA fused with genes to be inserted.…”