【正文】 ........................................1 微生物育種技術(shù)概述 ...................................................................................1 同源重組技術(shù)發(fā)展 .......................................................................................2 Red 同源重組技術(shù) .......................................................................................3 CRISPR 介導(dǎo)基因的編輯技術(shù) ............................................................................4 第二章 質(zhì)粒 ptarget 的構(gòu)建 ..................................................................................................8 1 材料 ..........................................................................................................................8 菌株和質(zhì)粒 .......................................................................................................8 培養(yǎng)基與常用溶液 ............................................................................................8 克隆中所用到的引物 .........................................................................................9 儀器 ................................................................................................................9 2 方法 ......................................................................................................................9 PCR 反應(yīng)體系與反應(yīng)條件 ............................................................................... 10 凝膠回收 ........................................................................................................ 10 gibson .......................................................................................................... 11 將質(zhì)粒 Ptarget 導(dǎo)入 DH5α中 ...................................................................... 11 ptarget 中 N20 的測序 ...................................................................................... 12 ptarget 質(zhì)粒提取 .............................................................................................. 12 3 結(jié)果分析 ............................................................................................................. 12 ptarget 質(zhì)粒的構(gòu)建 .......................................................................................... 12 N20 測序?qū)?比 ................................................................................................. 13 4 討論 ......................................................................................................................... 14 第三章 基因組片段的獲取與驗(yàn)證 ....................................................................................... 15 1 材料 ........................................................................................................................ 15 菌株和質(zhì)粒 .................................................................................................... 15 引物 .............................................................................................................. 15 大腸桿菌培養(yǎng)基 ............................................................................................. 16 儀器 .............................................................................................................. 16 2 方法 ........................................................................................................................ 16 細(xì)菌基因組的分離 .......................................................................................... 17 PCR 反應(yīng)體系與反應(yīng)條件 ............................................................................... 18 DNA片段的回收與純化 .................................................................................. 19 T 克隆的反應(yīng)體系 與反應(yīng)條件 ......................................................................... 19 化學(xué)轉(zhuǎn)化 ....................................................................................................... 20 T—clone vector 的菌落驗(yàn)證 PCR 反應(yīng)體系與反應(yīng)條件 ..................................... 20 3 結(jié)論與分析 ............................................................................................................. 20 muBL21 基因組 DNAtyrA、 tnaB、 talB、 gabD、 gabT 基因的驗(yàn)證 ................ 20 4 討論 ......................................................................................................................... 21 II 第四章 基因同源重組 ................................................................................................. 22 1 材料 ......................................................................................................................... 22 菌體和質(zhì)粒 ............................................................................................... 22 培養(yǎng)基與常用溶液 .......................................................................................... 22 常用溶液與試劑 ........................................................................................... 23 引物 ............................................................................................................... 23 儀器 .............................................................................................................. 23 2 方法 ........................................................................................................................ 24 wtBL21 感受態(tài)的制備 .............................................................................. 24 cas9 質(zhì)粒的電轉(zhuǎn)化 ..................................................................................... 24 wtBL21/cas9 感受態(tài)的制備 ............................................................................ 24 各基因片段的同源重組 .................................................................................. 25 重組驗(yàn)證菌落 PCR 的反應(yīng)體系與反應(yīng)條件 ..................................................... 25 同源重組成功的單克隆的獲取 ........................................................................ 26 Ptarget和 cas9 質(zhì)粒的去除 .............................................................................. 28 3 結(jié)果與分析 .......................................................................................................... 28 4．討論 .............................