Cloning, Sequence Modification And Functional Expression Of Structural Genes Of Bacillus Subtilis Biotin Operon

It was one of hot spots of high-technologic competition that biotin was produced by using genetically engineered technology. It could improve carcass quality meat quality, reproductive ability of livestock, and was indispensible matter for normal elaboration of livestock's varietal performance.Up till now, biotin could only be gained by the method of chemical synthesis and only imported from the foreign countries,and its price was very expensive and seriously restricted the development of our country's animal husbandry. Alongside rising of genetically engineered technology, the people begined to research biotin biosynthesis. The biosynthetic method could carry on the specific synthesis of biotin and overcome the low yield, nonspecific synthesis and nessary use of the chemical separation for chemically synthetic method.This method could reduce manufacturing cost of biotin, and improved economic benefits of animal husbandry. The use of this method had importantly theoretical meaning and economic value.Used As 1.1094 strain of Bacillus subtilis as researched material, the structural genes of biotin operon(bio operon) were cloned, and its sequence were engineered. Then the engineered genes of bio operon were carried on testing of the functional expression, and bioW & bioB gene were induced to express. This experiment probed how to improve the expression efficiency of the structural genes of bio operon, and provided the feasibly researched route and scientifically theoretical foundation. For highly efficient expression of the structural genes of bio operon, a lot of researched materials were constructed which could commercially operated in this experiment and these materials provided some explored experience and reference materials for future research. The study mainly carried on several research followed: 1. Extraction of large-fragment genomic DNAIn order to gain DNA template of PCR amplification (Long PCR amplification and salvage PCR amplification) which was high purity and large fragment, three methods were used to extract genomic DNA of Bacillus subtilis, i.e. low melting-point agarose embedding method, SDS-Proteinase K-Phenol Chloroform extraction method and bacterial genomic DNA extraction kit method. The genomic DNA of Bacillus subtilis were gainedby these methods, and the operated programs of the methods were improved. The results showed that the genomic DNA extracted by low melting-point agarose embedding method were obviously biggest than that of another two methods. Used the genomic DNA extracted by low melting-point agarose embedding method as PCR template, the full length of structural genes of Bacillus subtilis bio operon were gained by long PCR method. The results also explained that it was feasible that the genomic DNA of Bacillus subtilis were extracted and gained large DNA fragments by low melting-point agarose embedding method.2. Cloning of structural genes of Bacillus subtilis bio operonDiluted the genomic DNA of Bacillus subtilis as the template, long PCR product (10.3kb) and three salvage PCR products were separately gained by optimization of reaction conditions of PCR. Then used TA cloning method, The plasmids of three fragments( bio-1 bio-2 bio-3) of structural genes of Bacillus subtilis bio operon were gained by the purity of PCR products adding A in the two side of the products ligation with pGEM-T vector transformed Escherichia col identification and sequencing of recombinant plasmid. Contrasting analysis with the genes sequence of Bacillus subtilis 168 strain's bio operon, the sequence of bio operon of As 1.1094 strain had 12 bases difference with that of 168 train,and 7 bases caused variation of amino acid.Used long PCR primers as sequencing primer, the two sides of sequence of the long PCR product were separately sequenced one reaction by the direct sequencing method of PCR products, and the sequencing sequence were carried on constrasted analysis with sequence of 168 strain in Genebank. The results showed that the sequence of two strains basically coincided but...