A new lambda RES vector with a built-in Tn1721-encoded excision system

Abstract

A new lambda replacement vector for construction of genomic libraries was developed which allows the excision of cloned fragments by site-specific recombination from the lambda DNA and conversion into autonomously replicating plasmids. The vector system, derived from lambda EMBL4, is called lambda RES. It contains two recognition sites for site-specific recombination from Tn1721 on both sides of the replacement fragment of lambda EMBL4. Additionally, on one side, there is a plasmid replication origin from Rtsl with a kanamycin-resistance (KmR) marker. DNA fragments in the range of 8-14 kb may be inserted between BamHI or Sall sites in the lambda vector. Efficient excision and conversion of plaque-forming units into KmR colonies are obtained by infection of Escherichia coli strains harbouring Tn1739tnpR on a F' plasmid. Tn1739tnpR is a derivative of Tn1721 with a chloramphenicol-resistance-encoding gene (CmR), the lambda cI repressor gene, and a further copy of the resolvase-encoding tnpR gene under control of the tac promoter.