Stable Gene Duplication by “Puzzle” Shot Gun Cloning in Inverted Repeats of a Streptococcal Plasmid

Abstract

The amylase gene of Bacillus amyloliquefaciens was cloned by the usual shot gun method giving a highly expressed but unstable recombinant plasmid in Bacillus host cells. Stable recombinant plasmids were derived by recloning into the streptococcal plasmid pDBI01 (18 kb, 5–7 copies per cell) containing long inverted repeats.

For recloning we established a new variant of the shot gun technique termed the “puzzle” method. With this method, a frequently cutting restrictase (e.g., Sau3A) was used for both random linearization of the large vector plasmid molecules as well as partial digestion of the donor plasmid DNA. Thereafter, ligation of donor and vector DNA resulted in a heterologous population of recombinant plasmids harboring different donor fragments in different sites of the vector plasmid. With respect to the level of amylase production and plasmid stability all expected combinations were found: (1) low level - unstable; (2) low level - stable; (3) high level - unstable; and (4) high level - stable. In this way, ligation and selection are comparable with a puzzle and, for application, highly expressed and simultaneously stable recombinant plasmids can be considered as genetically engineered constructs of high fitness. In the case of insertion into the inverted repeats of pDB101, stability of recombinant plasmids was attained by spontaneous, recE independent duplication of the donor fragment containing the amylase gene. Such gene duplication gave a remarkable gene dosage effect with 2-fold amylase production at high expression level.