3] Restriction site bank vectors for cloning in gram-negative bacteria and yeast

Abstract

Publisher Summary This chapter describes the application of the restriction site banks for the construction of Escherichia coli vectors, wide-host-range cosmid vectors (able to grow in most gram-negative bacteria), and E. coli –yeast shuttle vectors. The chapter summarizes the restriction site bank series of vectors that have been constructed as tools for the genetic manipulation of DNA. The advantage of the restriction site bank vectors is that they maximize the choice of restriction enzymes that can be used for cloning and facilitate subsequent subcloning and deletion analysis. Thus, they permit, in a single plasmid, a variety of operations that could otherwise be done only with a combination of several different vector systems. Of the known restriction endonucleases that recognize 6-bp palindromes, 39 have unique recognition sites in E. coli vector pJRD184 and four others, while not unique, can also be used for cloning without disturbing essential vector functions. The example illustrated in the chapter involves the cloning of the genes for vanillate utilization from an uncharacterized pseudomonad ATCC 19151. Vanillate is a key intermediate in the degradation of the lignin molecule, one of the world's most abundant carbon sources and a potential source of industrial raw material and energy. This system is also a useful choice to demonstrate the generality of the cosmid cloning, conjugal transfer, and complementation method of gene isolation.