1 / 19

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

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

aleta
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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 Supernatant, pellet Read protein size then calculate the gene size

  7. Method—Cloning Technique • Insert：Chromosome DNA extractionPartial digest （Sau3AI）Gel extraction （500bp-2kb） • Vector：Puc19Digested by HindIIIAlkaline phosphotaseGel 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 sequenceAccession: 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!!

  19. The End Thank you for your attention

More Related