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 IntroductionXylanase, CMCase廢料的處理資源回收再利用農業畜牧業的利用BacillusCloning TechniqueMethodSample 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ZymogramCongo Red AssayXylan Congo red AcetateZymogramRead protein size then calculate the gene size Supernatant, pelletMethodCloning TechniqueInsert： Chromosome DNA extraction Partial digest （Sau3AI） Gel extraction （500bp-2kb）Vector： Puc19 Digested by HindIII Alkaline phosphotase Gel extractionpUC19Partial DigestionPuc19 digested by HindIIIMethodCloning TechniqueLigationElectroporesisLigationMethodClones AssayAmpicillin-IPTG-X-gal assay（Blue-white assay）Congo red assayPlasmid extraction, restriction enzyme checkZymogramPlasmid AssayDigested by PvyII, two cut sites on pUC19MethodFurther experimentSubcloningSequencingProtein purificationProtein activity assayCommercial applicationFurther researchResult32 xylanase active bacilli were isolated28 CMCasa active bacilli were isolatedNo xylanase or CMCase active clone was detectedDiscussionPartial digestAmpicillin affects congo red assayBlue-white assay efficiency is not so reliableThe reason of failureImmature operating!Bacillus anthracis str. KrugerB, unfinished sequence Accession: NC_004126 Number of Contigs: 887 Total Bases Sequenced: 10 mb The gene we are seeking: 500-2000 bpRate: get 1 positive clone in more than 10000 clones!The EndThank you for your attention