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.
Jang
S J
SJ
Kyung Hee University, Suwon, Korea.
Park
W J
WJ
Chung
S K
SK
Jeong
C Y
CY
Chung
D K
DK
D001483:Base Sequence; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014169:Transformation, Bacterial
D005819:Genetic Markers; C074775:CelA endoglucanase; D002480:Cellulase