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

2. 杜鎮  中研院植物所 425 室  學經歷：德國慕尼黑大學博士

3. 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

4. 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

5. Congo Red Assay  Xylan Congo red Acetate

6. Zymogram Read protein size then calculate the gene size Supernatant, pellet

7. Method—Cloning Technique  Insert ： Chromosome DNA extraction Partial digest （ Sau3AI ） Gel extraction （ 500bp-2kb ）  Vector ： Puc19 Digested by HindIII Alkaline phosphotase Gel extraction

8. pUC19

9. Partial Digestion

10. Puc19 digested by HindIII

11. Method—Cloning Technique  Ligation  Electroporesis

12. Ligation

13. Method—Clones Assay  Ampicillin-IPTG-X-gal assay （ Blue-white assay ）  Congo red assay  Plasmid extraction, restriction enzyme check  Zymogram

14. Plasmid Assay Digested by PvyII, two cut sites on pUC19

15. Method—Further experiment  Subcloning  Sequencing  Protein purification  Protein activity assay  Commercial application  Further research

16. Result  32 xylanase active bacilli were isolated  28 CMCasa active bacilli were isolated  No xylanase or CMCase active clone was detected

17. Discussion  Partial digest  Ampicillin affects congo red assay  Blue-white assay efficiency is not so reliable

18. The reason of failure  Immature operating!!  Bacillus anthracis str. KrugerB, unfinished sequence Accession: NC_004126 Number of Contigs: 887 Total Bases Sequenced: 10 mbNC_004126  The gene we are seeking: 500-2000 bp  Rate: get 1 positive clone in more than 10000 clones!!

19. The End Thank you for your attention