Towards single-copy gene expression systems making gene cloning physiologically relevant: lambda InCh, a simple Escherichia coli plasmid-chromosome shuttle system

Abstract

We describe a simple system for reversible, stable integration of plasmid-borne genes into the Escherichia coli chromosome. Most ordinary E. coli strains and a variety of pBR322-derived ampicillin-resistant plasmids can be used. A single genetic element, a lambda phage, is the only specialized vector required. The resultant strains have a single copy of the plasmid fragment inserted stably at the lambda attachment site on the chromosome, with nearly the entire lambda genome deleted.

FIG. 1

Plasmids on which the lambda InCh vectors are based. These are suitable cloning vectors for use with lambda InCh1. Both contain the Tac promoter from pKK223-3 (5).

FIG. 2

The three steps involved in transfer of an expression system from a pBR322-derived plasmid to the chromosome in stable single copy. At the upper left, the plasmid and the phage are shown as concentric circles with the phage outside. In the first step, recombination at both the 358-bp ′bla region and the 409-bp near-ori region results in transfer of the promoter gene expression system and a functional bla (Amp^r) gene to the phage replacing the Kan^r gene. In the second step a lysogen is formed by site-specific recombination at the att site, conferring ampicillin resistance and temperature sensitivity. The orientation at the att site is indicated relative to the flanking markers gal and bio. In the third step, recombination in the 800-bp direct-repeat region, near-att, removes most of the phage DNA conferring temperature independence. The att region in the stabilized strain is shown at the bottom.

FIG. 3

Localization of GFP-FtsI (top row) and GFP-FtsQ (bottom row). The GFP fusion proteins were expressed, without induction, from the chromosome by using lambda InCh (left column) or from plasmids (right column). Strains expressing GFP-FtsI and GFP-FtsQ from the chromosome are EC436 and EC442, respectively (6, 21), while cells expressing the proteins from plasmids are JOE426 and JOE427, respectively. Cells were grown at 30°C to early log phase and were fixed for fluorescence microscopy as described previously (6).