Abstract
A new λ replacement vector for construction of genomic libraries was developed which allows the excision of cloned fragments by site-specific recombination from the λ DNA and conversion into autonomously replicating plasmids. The vector system, derived from λEMB L4, is called λRES. It contains two recognition sites for site-specific recombination from Tn1721 on both sides of the replacement fragment of λEMBL4. Additionally, on one side, there is a plasmid replication origin from Rtsl with a kanamycin-resistance (Km R) marker. DNA fragments in the range of 8–14kb may be inserted between BamHI or SalI sites in the λ vector. Efficient excision and conversion of plaque-forming units into Km R colonies are obtained by infection of Escherichia coli strains harbouring Tl739 tnpR on a F′ plasmid. Tn l739tnpR is a derivative of Tn1721 with a chloramphenicol-resistance-encoding gene (Cm R), the λ cl repressor gene, and a further copy of the resolvase-encoding tnpR gene under control of the tac promoter.
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