Construction And Optimization Of E.coli And B.Subtilis Shuttle Vector

It is important that using shuttle vector in studying gene expression,gene function and mechanism expressing modulation 。Presently, different expression system has been constructed in E.coli,B .subtilis,Yeast,insect cell and mammalian cell. The characteristic and genetic background of E.coli has been studied clarity through long study, and B.subtilis is no harmful. So it is worth constructing Shuttle Vector of E.coli and B.subtilis to express gene. Used promoter P59 as promoter to construct a shuttle vector GJ01 with gene chloramphenicol as riddling gene, and bga was used as report gene to detect its express ability. In order to get a good vector , we optimize the vector through replace its RBS. Replicator of E.coli were cloned through PCR, and then connect with pBluSKM to get plasmid E3. Replicator and MCS from E3 were cloned to PCR amplification of pGDV1 to get pGDVM, which is a shuttle clone vector. There has two replicator ,gene chloramphenicol and MCS in pGDVM, it is easy to clone operation between E.coli and B.subtilis.Used promoter P59 as promoter and pGDVM as framework, get Ribosome Binding Site and terminator were got by PCR amplication from pAX01 were cloned to pGDVM , then got the shuttle vector GJ01. Use bga as report gene to detect its express ability, the highest activity of Bga in E.coli and B.subtilis were partly 75.3 and 83.2 miller, indicated it has high express ability. we optimize the vector through replace its RBS1 by RBS2, RBS3, RBS4 and RBS5, the result is that: activity of GJ02-bga in E.coli is 253.8 miller, and Activity of GJ05-bga in B.subtil is 135.4 miller. All this establish groundwork for construct expression system between E.coli and B. subtilis.Used B. subtilis 1094 as research material, the structural genes bioW and bioB were cloned, and its express vector GJ01-bioW and GJ01-bioB were constructed. Biotin express amounts were measured through animalcule and HPLC. The result is that Biotin express amounts of engineered B.bacilis is lower than that of wild B.bacilis.