NOVEL CLONING SYSTEM FOR DIRECT SCREENING USING A SUICIDAL STRATEGY

The pAC92 plasmid is a direct screening cloning vector which allows po sitive selection of recombinant clones (re-clones). This new high-copy -number plasmid vector encodes ampicillin resistance and carries the B acillus subtilis alpha-amylase (alpha-Amy)-encoding gene (amy) contain ing a multiple cloning site. The pAC92 plasmid confers to Escherichia coil transformants an amylolytic phenotype easily detected by iodine v apor staining. The re-clones are identified by insertional inactivatio n of alpha-Amy activity. During pAC92 construction, a bacterial growth defect was observed in host cells after some modifications of the pro moter region that caused the increase in the amy expression. This suic ide characteristic permitted the positive selection of re-clones. A se cond transformation step was performed to enhance the rate of re-clone s per plate.