Open AccessCloning and expression in Escherichia coli of dextranase genes from Bacteroides thetaiotaomicron
Author(s) -
Joncquiert J.C.,
Béchet M.,
Tierny Y.,
Courtois J.,
Dubourguier H.C.,
Guillaume J.B.
Publication year - 1991
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1991.tb04609.x
Subject(s) - dextranase , subcloning , pbr322 , insert (composites) , escherichia coli , microbiology and biotechnology , biology , clone (java method) , operon , cloning (programming) , bacteroides thetaiotaomicron , plasmid , expression vector , genomic library , molecular cloning , haeiii , restriction enzyme , recombinant dna , bacteroides , gene , gene expression , dextran , biochemistry , bacteria , genetics , polymerase chain reaction , mechanical engineering , computer science , restriction fragment length polymorphism , engineering , programming language , base sequence
A genomic bank was constructed in Escherichia coli HB101, consisting of DNA fragments from Bacteroides thetaiotaomicron strain 489 inserted within the vector pBR322. By screening on complex medium containing blue dextran, 10 stable dextranase‐positive (Dex + ) clones were isolated. Seven groups of Dex + inserts were identified on the basis of their restriction maps and hybridization responses. Dextranase activity of the recombinant clones was weak, and was revealed on the selection medium after 15 days. Subcloning of a Sau 3AI partially digested 3.2‐kb insert in the expression vector pDR720 greatly enhanced dextranase activity on blue dextran plates in one clone, but the delay remained unaltered. This suggested that the enzyme was released by cell lysis. Expression of this 0.7‐kb subcloned insert was dependent on the promoter region of tryptophan operon carried by pDR720.