Flora XM70 Metagenomic Library Construction And Cellulase Gene Cloning And Expression

Cellulase is widely used in food industry renewable energy production and other industrial production. Discovering strains with high cellulase activity and high efficient production is the continuous exploring issue. Only a few strains are on the stream currently, and there is still much room for improvement on their capacity and the cellulase’catalytic ability.This study chose a thermophilic cellulose-degrading bacterial flora reserved in our lab which enriched from a hot spring in Xiamen as the studied material used the meta genomic methods in the hope of discovering new cellulase genes. And then recombined the new gene into E.coil, to build a new high-yield cellulase engineered bacteria.Firstly, the total genomic DNA was extracted by CTAB/NaCl and evaluated the quality by AGE (agarose gel electrophoresis), which indicate the extracted DNA is satisfied the following meta genomic library construction experiment. The library’s construction used Copy Control Fosmid Library Production Kit made by Epicentre Biotechnologies and the operations was completely followed the kit’s working instruction. After the library’s building the researcher picked positive clones extracted the recombinant plasmid to testing and assessing the quality of library. It found that the inserted DNA fragment sizes were 30-40Kb and insert success rate up to 100%. Random restriction maps of recombinant plasmid were significantly different, which indicated that contains the gene were diversity. The restriction map of the first generation and 100 generation of plasmid DNA clones of the library was no significant difference which indicated that the library’s stability was very good and there were no insert fragment loss or rearrangement.On the basis of the preliminary sequencing results of the meta genome XM70 flora DNA, we designed primers to amplify cellulase gene from meta genome DNA, ligated the gene with expression vector pGEX-4T-2, transformed into host cells E. coil. After the confirmation that the clones get the right sequence, we used IPTG to induce the recombinant to a larger number of cellulase expression. The molecular weight of the recombinant protein was determined by SDS-PAGE. We tested the recombinant cellulase enzymatic properties using the crude enzyme solution which made from fermentation liquid by sonication. And the results indicate that the recombinant cellulase optimum temperature is 70℃, and the optimal pH is 7.0. In addition,the recombinant cellulase reaction temperature and pH range is wide.