Construction of glucanase gene of Bacillus subtilis in Escherichia coli
Subject Areas : Molecular MicrobiologyMarzieh Shavali 1 , Abbas Doosti 2
1 - M.Sc., Department of Biology, Faculty of Basic Sciences, Islamic Azad University, Shahrekord branch, Shahrekord, Iran.
2 - Associate Professor, Biotechnology Research Center, Shahrekord branch, Islamic Azad University, Shahrekord, Iran.
Keywords: Bacillus subtilis, β-glucanase, T / A Cloning,
Abstract :
Background & Objectives: The grains are considered as the main food for poultries. Grains contain significant content of non- soluble starch polysaccharides (NSP). NSPs cause negative impact and incompatibility in birds. Digestion of NSP contents by β-glucanase enzyme supplement reduces the viscosity of polysaccharides in the digestive tracts, and increases their intestinal absorption. The aim of this study was to construction of glucanase gene of Bacillus subtilis in Escherichia coli. Materials & Methods: In this experimental study, the glucanase gene of B. subtilis was amplified using specific primers and polymerase chain reaction. After purification of the bands, Bgl gene was cloned by T/A cloning technique in pGEM vector, and was transformed in E. coli. Afterward, the pGEM-bgl was transferred into E. coli Top-10 strain. The recombinant vectors were then transformed into E. coli competent cells, and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The recombinant plasmid transformed to E. coli Top10 cell and the colonies carrying plasmid were selected by PCR. The presence of glucanase construction was confirmed by enzymatic digestion. Results: The results showed that the glucanase gene was successfully cloned in E. coli. The results of cloning of 767 bp glucanase gene was confirmed by PCR assay. Conclusion: The construction of B. subtilils glucanase gene and cloning of this gene in E. coli is a strong alternative for the production of probiotic used for poultry.