Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers

Abstract

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

Expression and characterization of a structural and functional domain of the mannitol-specific transport protein involved in the coupling of mannitol transport and phosphorylation in the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli

“…The parental vector pMcCIIIi showed only the expected 1.4-kb fragment (Figure 1). Subcloning of the HindIII fragment containing IIBmfl, fused to the ribosomal binding site (RBS) and ATG start codon of the cro gene downstream of the PR promoter, was done into the parental phagemid pMc5-8 (Stanssens et al, 1989) to delete the A domain. The HindIII fragments were excised from vector pMcCIIIam using HindIII restriction enzyme, gel-purified, and ligated into the vector pMc5-8 linearized with the same enzymes.…”

Fine tuning of the catalytic properties of human carbonic anhydrase II

“…After transfection, the mutated human carbonic anhydrases I1 were expressed in the lonEscherichia coli strain SG20043. To make the Thr200-tArg variant, we used the plasmid pACA constructed in the laboratory of C. Fierke (Duke University) and consisting of the human carbonic anhydrase I1 gene [lo] behind a T7 RNA polymerase promoter [ll] in the pMa/c vector [12]. Singlestranded DNA was prepared from pACA using the helper phage M13K07 [12].…”

“…To make the Thr200-tArg variant, we used the plasmid pACA constructed in the laboratory of C. Fierke (Duke University) and consisting of the human carbonic anhydrase I1 gene [lo] behind a T7 RNA polymerase promoter [ll] in the pMa/c vector [12]. Singlestranded DNA was prepared from pACA using the helper phage M13K07 [12]. Mutagenesis was performed as described above using single-stranded DNA from pACA and a 20-mer synthetic oligonucleotide.…”

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

“…Genetic manipulations pMA5-8pL and pMC5-8pL, derivatives of pMA5-8 and pMC5-8 carrying the pL promoter (Klippel et al, 1988), were used for site-directed mutagenesis of the Opc protein through alternate strand selection by means of the pMAC plasmid vector system (Stanssens et al, 1989). The coding region of the opc gene was amplified from plasmid pBE501 (Olyhoek et al, 1991) using oligonucleotides opcAvr II (5Ј-GCG ACC CCT AGG AGA TTA ACT ATG AAA AAA ACA G-3Ј, position 380-398, mutated nucleotides underlined, Avr II site bold) and opcHin dIII (5Ј-CAC GTG AAG CTT AAC CGC GTA TTT TAA CCG-3Ј, position 1329-1346).…”