Mark Vasser

Cassette mutagenesis : an efficient method for generation of multiple mutations at defined sites

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.

CONSTRUCTION OF THREE HYBRID PROMOTERS AND THEIR PROPERTIES IN ESCHERICHIA COLI

SUMMARY This paper describes three hybrid promoters which are functional in Escherichia coli . In the case of the first hybrid promoter ( tac I) sequences upstream of position −20 were derived from the trp promoter and sequences downstream of position -20 were derived from the lac -UV5 promoter. This hybrid promoter is seven times stronger than the lac -UV5 promoter. It can be repressed by the lac-repressor and induced by isopropyl-β-D-thiogalactoside (IPTG). In the case of second hybrid promoter ( tac II), we used the DNA sequences upstream of the Hpa I site (which is located in the Pribnow box of the trp -promoter) and fused those sequences to a synthetic DNA fragment of 46 bp. The sequence of the synthetic fragment creates a new Pribnow-box which is followed by the lac -operator. Downstream from the lac -operator are nucleotides that code for a Shine-Dalgarno (SD) sequence. The Shine-Dalgarno sequence is flanked by two restriction sites which allows us to exchange different Shine-Dalgarno sequences. Thus, we constructed an inducible promoter with a portable Shine Dalgarno sequence which forms an active ribosome binding site when fused to the start codon of a foreign gene. The tac II promoter is as efficient as the tac I promoter. The third hybrid promoter (rac 5-16) is a hybrid between the rrnB promoter and the lac UV5 promoter. Its structure resembles that of the tac I promoter. At the junction, in the area of -20, three unique restriction sites were introduced. This makes it possible to change the distance and the nucleotide sequence between the –35 area and the −10 area (the Pribnow-box).