土壤菌Bacillus之分离和木聚糖水解脢及纤维素水解脢活性分析.pptVIP

土壤菌Bacillus之分離與木聚糖水解脢及纖維素水解脢活性分析 學 生：何季庭 指導教授：杜 鎮 研究員 杜鎮 中研院植物所425室 學經歷：德國慕尼黑大學博士 Introduction Xylanase, CMCase 廢料的處理 資源回收再利用 農業畜牧業的利用 Bacillus Cloning Technique Method—Sample preparation 土壤樣品的採取 Bacillus分離 單株分離 Congo red assay多糖類水解脢活性分析 Zymogram Congo Red Assay Xylan Congo red Acetate Zymogram Method—Cloning Technique Insert：Chromosome DNA extractionPartial digest （Sau3AI）Gel extraction （500bp-2kb） Vector：Puc19Digested by HindIIIAlkaline phosphotaseGel extraction pUC19 Partial Digestion Puc19 digested by HindIII Method—Cloning Technique Ligation Electroporesis Ligation Method—Clones Assay Ampicillin-IPTG-X-gal assay（Blue-white assay） Congo red assay Plasmid extraction, restriction enzyme check Zymogram Plasmid Assay Method—Further experiment Subcloning Sequencing Protein purification Protein activity assay Commercial application Further research Result 32 xylanase active bacilli were isolated 28 CMCasa active bacilli were isolated No xylanase or CMCase active clone was detected Discussion Partial digest Ampicillin affects congo red assay Blue-white assay efficiency is not so reliable The reason of failure Immature operating!! Bacillus anthracis str. KrugerB, unfinished sequence Accession: NC_004126 Number of Contigs: 887Total Bases Sequenced: 10 mb The gene we are seeking: 500-2000 bp Rate: get 1 positive clone in more than 10000 clones!! The End Thank you for your attention * * Read protein size then calculate the gene size Supernatant, pellet Digested by PvyII, two cut sites on pUC19