Construction and Characterization of a Highly Regulable Expression Vector, pLAC11, and Its Multipurpose Derivatives, pLAC22 and pLAC33

Abstract

A number of different expression vectors have been developed to facilitate the regulated overproduction of proteins in Escherichia coli and related bacteria. Some of the more popular ones include pKK223-3, pKK233-2, pTrc99A, and the pET family of expression vectors. These vectors were designed to be regulable and can be grown under conditions that repress protein production or under conditions that induce protein production. Unfortunately, however, numerous researchers have found that these vectors produce significant amounts of protein even when grown under repressed conditions. We describe here a new expression vector, pLAC11, which was designed to be more regulable and thus more tightly repressible when grown under repressed conditions. The tight regulation of pLAC11 was achieved by utilizing the O3 auxiliary operator, CAP binding site, promoter, and O1 operator that occur in the wild-type lac control region. The pLAC11 vector can be used to conduct physiologically relevant studies in which the cloned gene is expressed at levels comparable to that obtainable from the chromosomal copy of the gene in question. In experiments in which a bacterial cell contained both a null allele in the chromosome and a second copy of the wild-type allele on pLAC11, we observed that cells grown under repressed conditions exhibited the null phenotype while cells grown under induced conditions exhibited the wild-type phenotype. Two multipurpose derivatives of pLAC11, pLAC22, and pLAC33 have also been constructed to fulfill different experimental needs.

The PduM Protein Is a Structural Component of the Microcompartments Involved in Coenzyme B12-Dependent 1,2-Propanediol Degradation by Salmonella enterica

“…Studies were done to determine whether the pduM deletion mutant could be complemented by pduM expressed from a plasmid. The vector used for complementation was pLAC22, which allows tight regulation of gene expression by IPTG (59). The results presented in Fig.…”

“…For complementation studies, the coding sequences of pduM were cloned into pLAC22 via PCR with template pEM55 as previously described (9). Vector pLAC22 allows tight regulation of protein production by IPTG (59). The DNA sequence was verified.…”

PduL Is an Evolutionarily Distinct Phosphotransacylase Involved in B 12 -Dependent 1,2-Propanediol Degradation by Salmonella enterica Serovar Typhimurium LT2

“…The primers used for amplification were 5Ј-GCCGCCAGATCTATGGATAAAGA GCTTCTGCAATCA-3Ј and 5Ј-GCCGCCAAGCTTATTATCGCGGGCCTAC CAGCCG-3Ј. These PCR primers introduced BglII and HindIII restriction sites that were used for cloning into vector pLAC22 (39). Following ligation, clones were introduced into Escherichia coli DH5␣ by electroporation, and transformants were selected by plating onto LB agar supplemented with 100 g ml Ϫ1 ampicillin (Amp) (18).…”

Purification and Initial Characterization of the Salmonella enterica PduO ATP:Cob(I)alamin Adenosyltransferase

“…The DNA sequence of each clone was verified, and the resulting strains (BE294 to BE298) were used for production of truncated versions of the PduO enzyme.For complementation studies, truncated versions of the pduO gene were subcloned from the expression vectors into pLAC22 by using BglII and HindIII restriction sites. Vector pLAC22 allows the expression of cloned genes to be very stringently regulated (29). The resulting plasmids were used to transform S. enterica TR6579 by electroporation.…”