pUC Vectors

pUC Vectors - Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial. preparation i.e. University of California). These plasmids are 2,700 bp long and possess,

(i)ampicillin resistance gene,

(ii)the origin of replication derived from pBR322 and

(iii)the lac Z gene derived from E. coli. Within the lac region is also found a polylinker sequence having unique restriction sites (identical to those found in phage M13. When DNA fragments are cloned in this region of pUC, the lac gene is inactivated.

These plasmids when transformed into an appropriate E. coli strain, which is lac (e.g. JM103, JMI09), and grown in the presence of IPTG (isopropyl thiogalactoside, which behaves like lactose, and induces the synthesis of f3 galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.

On the other hand, pUC having no inserts and transformed into bacteria will have an active lac Z gene and therefore will produce blue colonies, thus permitting Identification of colonies having pUC vector with cloned DNA segments.

The cloning vectors belonging to pUC family arc available in pairs with reversed orders of restriction sites relative to lac Z promoter. pUC8 and PUC9 make one such pair . Other similar pairs include pUC12 and pUCl3 or pUC18 and pUC19.

As discussed above, in pBR322 and pBR327, the DNA is inserted at a site located in one of the two genes for resistance against antibiotics, so that it will inactivate one of the two resistance genes. The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and by their failure to grow in a medium containing both the antibiotics.

The plasmids carrying no insert on the other hand, will be able to grow in media containing one or both the antibiotics. In this manner, the presence of lac Z gene in pUC and resistance genes against ampicillin and tetracycline in pBR322 and pBR327 allow selection of E. coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.