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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

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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

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  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

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