Plasmid-free clone of E. coli strain DSM 6601 and method for its creation using sacB and two resistance genes introduced into the pMut1 and pMut2 plasmids

摘要

Disclosed is a plasmid free clone of the E. coli strain DSM 6601, and a method of preparing it. The method involves the introduction of a resistance gene and the sacB gene into each of the plasmids pMut1 and pMut2, which are the plasmids that are usually found in this strain. The modified plasmids are introduced into the bacteria under conditions such that the naturally occurring plasmids are replaced by the modified ones. The bacteria are grown under conditions selecting for the particular resistance used in the plasmids, and then are grown under conditions that select against sacB. Preferably each plasmid has a different antibiotic resistance, and more preferably the pMut1 plasmid has a tetracycline resistance cassette, and pMut2 plasmid has a kanamycin resistance cassette, with the elimination of each plasmid taking place in two separate steps.