Development Of A Multifunctional Escherichia coli/Bacillus subtilis Shuttle Expression Plasmid With Synthetic Expression Control

Abstract

To simplify the screening of cloned DNA and to enhance the expression of protein products, a plasmid, pSP 10, was designed and constructed containing several features. These features included: 1) an origin of replication for Escherichia coli and Bacillus subtilis. 2) a selection gene, 3) an indicator gene, 4) a cloning region, and 5) a region of expression control. The plasmid shuttle vector, pSP 10, contains a synthetic promoter sequence, which was used to direct the expression of the Lac Z gene. The Lac Z gene codes for the protein B-Galactosidase. Two secretion leader sequences were also constructed and cloned into the plasmid pUC 19 for characterization. They were labelled "PEN I" and "PEN II". When properly inserted upstream of (read before) a protein-coding sequence, the secretion leader would direct secretion of the translated protein across a cell membrane in either E. coli or EL gubfilig. The project involved growing the cells and purifying the pSP 10 plasmid DNA, analyzing the DNA for the synthetic promoter sequence, and selecting restriction sites in pSP 10 which would cut the plasmid to accept the secretion leader sequence. Analysis of the plasmids, pUC/PEN I and pUC/PEN II, by restriction endonuclease mapping resulted in the identification of restriction sites compatible with cloning PEN II into pSP 10.