Molecular Cloning in Bacillus subtilis

This chapter focuses on molecular cloning in Bacillus subtilis. Many bacilli can be grown to very high yield on inexpensive media. They are largely innocuous to humans and to plants and animals of commerical importance. The potentially most important property of the bacilli for commercial exploitation is their ability to secrete protein products into the culture medium. These products often accumulate to very high yield and can be recovered simply and inexpensively. B. subtilis has been one of the principal organisms used for the investigation of genetic transformation. However, protoplast transformation is limited in its usefulness by various properties: (1) it requires the use of complex media for protoplast regeneration and therefore, does not permit the direct selection of nutritional markers, (2) the efficiency of protoplast transformation has a very steep inverse dependence on DNA molecular weight; ligated (concatameric) plasmid DNA transforms poorly, and (3) the procedure is fairly laborious, and protoplasts, unlike competent cells, cannot be stored.