A METHOD FOR CONSTRUCTION OF ESCHERICHIA-COLI STRAINS WITH MULTIPLE DNA INSERTIONS IN THE CHROMOSOME

A system for construction of E. coli strains with multiple DNA inserti ons in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage lambda, has been developed. Circular non-replicating DNA fragments containing the transposon attachment si te (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strai n was obtained by insertion of the fragment containing transposon int- Tn gene coding for Int-Tn into the chromosome). Integration of these f ragments into the chromosome of int-Tn(+). cells gives rise to a colle ction of antibiotic-resistant clones with single insertions at differe nt locations in the chromosome. These insertions are transferred subse quently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage lambda. xis and int genes is introduced into the cells to excise the drug resista nce gene flanked with the lambda attL and lambda attR sites from the c hromosome. Cells cured of the helper plasmid can undergo the next cycl e of P1 transduction/drug resistance gene excision. Each cycle adds an other chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.