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BacillusCloning 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 extractionPartial digest （ Sau3AI ）Gel extraction （ 500bp-2kb ）

Vector ：Puc19Digested by HindIIIAlkaline phosphotaseGel 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 chec

k Zymogram

Plasmid AssayDigested 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 d

etected

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, unfinishe

d sequence Accession: 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 1000

0 clones!!

The EndThank you for your

attention