土壤菌Bacillus之分离与木聚糖水解脢及纤维素水解脢活性分析

1、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 AcetateZymogram Read protein size then c

2、alculate the gene size Supernatant, pelletMethodCloning TechniqueInsert：Chromosome DNA extractionPartial digest （Sau3AI）Gel extraction （500bp-2kb）Vector：Puc19Digested by HindIIIAlkaline phosphotaseGel extractionpUC19Partial DigestionPuc19 digested by HindIIIMethodCloning TechniqueLigationElectropore

3、sisLigationMethodClones 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 applicat

4、ionFurther 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: 887Total Bases Sequenced: 10 mb The gene we are seeking: 500-2000 bpRate: get 1 posi