Positive selection system for identification of recombinants using alpha-complementation plasmids.
A number of selection systems have been developed for direct selection of recombinant plasmids in cloning experiments (positive selection). In this study, the Commonly used LacZ-based alpha-complementation plasmid vectors have been used for designing a positive selection system for the selection of recombinants. The basis for the Strategy is the phenomenon of galactose sensitivity exhibited by galactose epimerase (galE) mutants of Escherichia coli. It is known that lacZ+ galE, but not lacZ- galE cells are killed upon addition of lactose due to the accumulation of a toxic intermediate, UDP-galactose, by hydrolysis of lactose. Using a galE mutant strain of E. coli that carries the lacZAM15 allele, various alpha-complementation plasmids that vary in their copy number were examined for their ability to be killed following addition of lactose. The results show that some plasmids that exhibit relatively high beta-galactosidase enzyme activity can be used effectively for positive selection. This selection would be extremely useful during primary cloning experiments such as construction of genomic or cDNA libraries and also in instances involving selection for rare recombinants.
Reddy
Manjula
M
Centre for Cellular and Molecular Biology, Hyderabad, India. manjula@ccmb.res.in
D003001:Cloning, Molecular / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic / Q000235:genetics*
D004269:DNA, Bacterial