Development of an expression strategy using a lytic phage to trigger explosive plasmid amplification and gene expression

Daniel J. O'Sullivan, Shirley A. Walker, Sandra G. West, Todd R. Klaenhammer

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

A novel plasmid-based expression strategy, exploiting two features of lytic bacteriophages, was developed in Lactococcus lactis. Components of this system include a phage origin of replication and phage expression signals, which were induced to high efficiency upon phage infection of the host. Phage-specific expression signals were cloned from φ31 in a promoter-screening strategy using the lacZ gene from Streptococcus thermophilus. One clone exhibited a significant induction in β-galactosidase production and concomitant increase in lacZ mRNA during the φ31 infection cycle of the host. Molecular characterization of the cloned insert revealed 888 bp positioned near the φ31 cos site. Primer extension analysis showed that transcription was induced ∼20 min following φ31 infection at four points, apparently organized in two sets of tandem promoters on the cloned phage insert. One of these middle phage promoters also showed a basal level of activity prior to phage infection. The φ31 promoter lacZ cassette was cloned into a low-copy-number vector plasmid containing the φ31 origin of replication (ori31) and the resulting low-copy-number plasmid exhibited negligible β-galactosidase production in L. lactis. However, >2,000 units were detected following a deliberate infection with φ31. A control expression plasmid without ori31 could only be induced to 85 units. The combination of these phage-inducible expression signals together with ori31 functioned synergistically to drive rapid and high efficiency expression of a heterologous gene in L. lactis.

Original languageEnglish (US)
Pages (from-to)82-87
Number of pages6
JournalBio/Technology
Volume14
Issue number1
DOIs
StatePublished - Jan 1996

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