Preparation and ligand screening of a lambda gt11 lysogen library.

Abstract

The λgt11 system has been designed to allow expression of cDNAs either in phage plaques or in bacteria lysogenized with recombinant λgtll phages (, ). cDNAs are cloned into a unique EcoRl restriction site near the 3′ end of a truncated E. coli lactose operon (lacP/O-lacZ), allowing inducible, high-level transcription of lacZ-cDNA-encoded fusion mRNAs that can be translated into fusion proteins if the cDNA coding strand is properly oriented with respect to lac transcription, and in phase with the p-galactosidase reading frame. A host strain is chosen that carries a lacl q allele on a high-copy number plasmid (pMC9), assuring efficient repression of lac transcription in the absence of an inducer. Since λgtll encodes a temperature-sensitive cl repressor (c/857), it can be lysogenized and stably maintained as a lysogen (i.e., as a “single copy vector”) at the permissive temperature, and subsequently made to switch to a “multi copy vector” on inducing lytic growth of the prophage by a temperature shift. An amber mutation in the S gene (SlOO&mb) renders the phage lysis-defective in a nonsuppressor host, allowing the accumulation of phage-encoded products inside the bacterial host cells. To maximize stability of lacZ- cDNA-encoded fusion protein, a host strain is used that carries a deletion in the gene for the Ion protease. λgtl 1 can also form plaques if grown on a host that carries an amber suppressor mutation (for a recent review on the λgtll system, see ref. )