| Biotin (vitamin H) is a sulfur-containing nature vitamin in B family, and is essential for animal to maintain the normal physiological function. By the appearance of the short of symptom in animal aquaculture, there is an new recognization of nutritional and physiological function of biotin, meanwhile, it has been one of the most significant vitamin in water soluble vitamin. Biotin is extensive used in animal husbandry,molecule biology,biofermentation and medicine field. Biotin was not directely obtained from naturae and was needed import from Japan or American every year, and cost expensively, so it is necessary to research the synthesis of biotin. Recently, it was one of hot spots of high-technologic competition that biotin was produced by using genetically engineered technology.Bacillus subtilis is harmless to humans and animals, is the best-characterized gram-positive bacterium known today on genetic, biochemical and physiological level, and can secret a large of industrial important enzymes into the medium. There are two factors influence the expressional efficiency of the gene: the strength of the promoter and the copies of gene, the low efficiencies of cloning and high levels of replicating instability in Bacillus subtilis, Integration of cloned genes into the chromosome is a strategy used to avoid genetic instability. Therefore, used B.subtilis 1A747 as researched material, the structural genes of biotin operon(bio operon) were cloned, integrated the strong promoter of Pâ–³glv and Cm resistance in Bacillus subtilis, increase the copies of biotin biosynthesis gene, identification and characterization its expression and function.Biotin biosynthesis operon (bioWAFDB) of Bacillus subtilis were cloned through PCR, and then connect with PGEM-T to get plasmid YG50, by the homology recombination (single crossover), the whole plasmid YG50 is integrated in 1A747 chromosome, named BI1. Respectively, used five primers, intedia-1, intedia-2, bioW-bioB, bioB-bioW, Spec-bioF, to...
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