ISSN:
1432-0614
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
Notizen:
Summary The recombinant phage λG1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo-β-1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations. The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene. The β-glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but β-glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the β-glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in β-glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Apr (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF00252158
