Abstract
This chapter discusses the use of pUB110 as a vector for cloning deoxyribonucleic acid (DNA) fragments in Bacillus subtilis (B. subtilis). Several plasmids have been tested for cloning; among these, pUB110 offers certain advantages, including relatively high transforming activity, stable maintenance in B. subtilis and related bacteria, high copy number, and sensitivity to several well-characterized endonucleases. The replication of pUB110 has been shown to occur in temperature-sensitive DNA replication mutants of B. subtilis at temperatures that are nonpermissive for chromosome replication, thereby indicating that this system may be useful for the amplification of cloned fragments. Several trp derivatives of pUB110 can be used for cloning HindIII-generated DNA fragments by screening for the insertional inactivation of plasmid-specified trpC-complementing activity. The methods used in the study, which is discussed in the chapter, reflect a direct extrapolation of principles established in the Escherichia coli (E. coli) systems, and no unique problems have been detected. Of particular interest is the absence of the obvious problem of restriction of foreign DNA fragments by B. subtilis because it has been reported that B. subtilis possesses a restriction system, which may cause difficulties in cloning DNA from sources other than Bacillus.