Minimal size plasmids containing an M13 origin for production of single-strand transducing particles

Abstract

We have studied the requirements for efficient production of single-strand transducing particles from minimal size plasmids containing the phage M13 origin of replication. The most favorable origin fragment for production of transducing particles was found to extend from nucleotides 5372 to 5943 of the phage sequence, which includes a segment of the phage gene IV coding region and eliminates part of the gene II promoter. Minimizing vector size for M13 origin plasmids appears to be beneficial since transducing particle titer decreases with increasing plasmid size. A mutant helper phage was isolated and shown to improve the production of transducing particles from small plasmids, but to outcompete plasmids bearing large inserts. Several plasmid vectors which produce high yields of single-strand transducing particles and good ratios of transducing particles to helper phage have been constructed.