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  Zymogram

Congo Red Assay  Xylan Congo red Acetate

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

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

pUC19

Partial Digestion

Puc19 digested by HindIII

Method—Cloning Technique  Ligation  Electroporesis

Ligation

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

Plasmid Assay Digested by PvyII, two cut sites on pUC19

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

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

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

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

The End Thank you for your attention