New E. coli cloning vector using a cellulase gene (celA) as a screening marker.

The extracellular endoglucanase A gene of Clostridium thermocellum (celA) was used as a screening marker for E. coli cloning vector A 1.4-kb EcoRI fragment containing celA from pTvec/celA was isolated and cloned into a pUC18 deleting beta-galactosidase gene fragment. The constructed vectors, pCEL1, pCEL10, pCEL11, and pCEL20, have different multiple cloning sites within celA. If the cellulase, CelA, is inactivated by insertion of a foreign DNA fragment into multiple cloning sites, the recombinant transformants show no clear halos on an agar plate containing cellulose. This process overcomes the ambiguity of color screening in the X-gal/beta-galactosidase system, and over 90% of the recombinant transformants with no halos have foreign DNA inserts. Several E. coli strains were transformed successfully with pCEL series vectors regardless of mutation for alpha-complementation. Because E. coli strains do not have a cellulase gene, a vector using a cellulase gene screening marker can be used in any E. coli strain without limit. The new cloning system is very efficient, convenient, and cost effective.