【正文】 畢 業(yè) 論 文 設計題目 用同源重組的方法進行基因修復 I 目錄 摘要 .....................................................................................................................................i Abstract................................................................................................................................ ii 第一章 同源重組技術的研究進展 .........................................................................................1 1 前言 ..........................................................................................................................1 微生物育種技術概述 ...................................................................................1 同源重組技術發(fā)展 .......................................................................................2 Red 同源重組技術 .......................................................................................3 CRISPR 介導基因的編輯技術 ............................................................................4 第二章 質粒 ptarget 的構建 ..................................................................................................8 1 材料 ..........................................................................................................................8 菌株和質粒 .......................................................................................................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 反應體系與反應條件 ............................................................................... 10 凝膠回收 ........................................................................................................ 10 gibson .......................................................................................................... 11 將質粒 Ptarget 導入 DH5α中 ...................................................................... 11 ptarget 中 N20 的測序 ...................................................................................... 12 ptarget 質粒提取 .............................................................................................. 12 3 結果分析 ............................................................................................................. 12 ptarget 質粒的構建 .......................................................................................... 12 N20 測序對 比 ................................................................................................. 13 4 討論 ......................................................................................................................... 14 第三章 基因組片段的獲取與驗證 ....................................................................................... 15 1 材料 ........................................................................................................................ 15 菌株和質粒 .................................................................................................... 15 引物 .............................................................................................................. 15 大腸桿菌培養(yǎng)基 ............................................................................................. 16 儀器 .............................................................................................................. 16 2 方法 ........................................................................................................................ 16 細菌基因組的分離 .......................................................................................... 17 PCR 反應體系與反應條件 ............................................................................... 18 DNA片段的回收與純化 .................................................................................. 19 T 克隆的反應體系 與反應條件 ......................................................................... 19 化學轉化 ....................................................................................................... 20 T—clone vector 的菌落驗證 PCR 反應體系與反應條件 ..................................... 20 3 結論與分析 ............................................................................................................. 20 muBL21 基因組 DNAtyrA、 tnaB、 talB、 gabD、 gabT 基因的驗證 ................ 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 質粒的電轉化 ..................................................................................... 24 wtBL21/cas9 感受態(tài)的制備 .....................................................................