Study On Cloning And Functional Expression Of Biotin Operon Genes In B.subtilis

Two DNA fragments, which are about 700bps and contain ori and rep of E.coli, were cloned from pUC19 using two pairs of primers respectively. Three E.coli-B.subtilis shuttle vectors pGDVM,pGDVM1 and pGDVM2whith chloramphenicol resistance, were then constructed with the framework pGDV1 , a plasmid come from B.subtilis strain 1E60. It indicates that the cloned 700 bp DNA fragments contains ori and rep of E.coli have a complete ability of replication, and the three shuttle vectors can inherit and clone steadily via continuous culture and times of shuttle experiments between E. coli and B. subtilis, This series of shuttle vectors based on plasmid pGDV1 , are numerous to 150-200 copies in B. subtilis . The length of vectors are only about 3kb ,they contain multiple clone site and chloramphenicol resistance gene,can act as ideal framework for B.subtis genetic engineering.Biotin operon genes bioB,bioW and gene cluster bioWAFDB in B. subtilis have been cloned and inserted into the downstream of Ptac promoter of E.coli expression vector pEXT20, and yield induced expression vectors —pEXT20bioW, pEXT20bioB and pEXT20bioWAFDB . Five E. coli mutant strains, R874,R875,R876,R877 and R879 with defect of bioF, bioB, bioC,bioD and bioA respectively, were used for genetic complement analysis of biotin genes. The results show that four biotin biosynthetic genes bioF, bioB, bioD and bioA in B. subtilis can express in E. coli, but the expression vector of bioW can not competent the bioC gene defect Without supplement of PADK. Inducing of IPTG under high level, the growth of host strains of pEXT20bioWAFD is inhibited. The growth inhibition results from the bioW gene but not from the overexpression of bioB.