Expression systems for anaerobic rumen bacteria gene cloning
1993
Gasparic, A. | Nekrep, F.V. (Biotechnical Faculty, Ljubljana (Slovenia). Zootechnical Dept.)
By comparative experiments concerning the desired host-expression systems characteristics, three of them: E. coli, B. subtilis and B. brevis 47-5 were tested. An expression system for cloning DNA fragments of anaerobic rumen bacteria coding for cellulases was selected. Chromosomal DNA sequence of Bacteroides succinogenes BL2 coding for endoglucanase was insert in pNU200 plasmid in two orientations and used for B. subtilis MT119 transformation. Using different B. subtilis and E. coli strains we bypassed several problems connected with molecular genetics of anaerobic microorganisms. With recombinant vectors - cloning and expression plasmids pIL253 and pNU200 we tried to test a high secretory potential of B. brevis 47-5. Ruminococcus flavefaciens chromosomal DNA sequence CMC P11 coding for endoglucanase was inserted in two orientations in plasmid pKK223-3. With this recombinant plasmid mixture the E. coli JM105 strain was transformed. The expression level was estimated in relation to insert orientation and different promoter usage
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