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摘要: 在完成了对各种微生物基因组的测序以后，功能基因学的研究变得尤为重要。研究基因功能最直接的方法便是将待研究的基因失活。快速、高效删除大肠杆菌染色DNA的目的基因是大肠杆菌代谢工程研究的前提和基础。本实验旨在利用Red重组系统和Xer重组系统获得大肠杆菌MG1655的LacY乳糖通透酶基因删除菌株。实验采用PCR扩增lacY基因片段，然后构建pUC-lacY载体，aflⅡ酶切并经T4 DNA聚合酶补平粘性末端后与dif-Gm-dif片段连接，得到重组质粒T-lacY∷dif-Gm-dif；PCR扩增获得突变盒lacY∷dif-Gm-dif片段，电转化突变盒片段入大肠杆菌细胞；PCR扩增验证是否获得没有抗性标记的ΔlacY基因删除菌株。本实验获得了重组质粒T-lacY∷dif-Gm-dif，化学转化法突变盒片段入大肠杆菌细胞获得的重组菌落，用验证引物3和引物4进行菌落PCR扩增，电泳验证不是阳性转化子，最后分析总结了一些影响基因敲除的因素。43490
毕业论文关键词: Red 重组系统；Xer 重组系统；lacY；dif-Gm-dif 片段；基因删除
Deletion of the lactose permease gene lacY in Escherichia coli Chromosome
Abstract：Since many DNA sequencing projects of varied microorganisms have been completed ,studies on their functional genomics become more important. Inactivation of an interesting gene is a direct method to characterize itsfunction. Rapid and efficient inactivation of the target gene on Escherichia coli chromosome is the precondition and groundwork of researches on metabolic engineering. It was to obtain the lacY deleted strain of E. coli MG1655 by using both Red and Xer recombination system. The sequence of lacY from E. coli MG1655 was first amplified by PCR then inserted into pUCm-T vector to obtain pUC- lacY. This vector was then digested by aflⅡ and T4 DNA polymerase was used to create blunt ends after digestion, following ligation with dif-Gm-dif fragment to form T-lacY∷dif-Gm-dif recombinant. lacY∷dif-Gm-dif was then amplified by PCR and transformed into E. coli by electroporation, following PCR analysis to confirm the designate ΔlacY strain of E. coli without antibiotic resistance gene was obtained. Results showed the recombinant vector T-lacY∷difGm was successfully constructed using a process mediated by Red and Xer recombination system. lacY gene was deleted by homologous recombination and selectable marker was removed from experiment strain, which was confirmed by PCR.
Keywords: Red recombination system； Xer recombination system； lacY； dif-Gm-dif fragmen； Gene deletion
目 录
1 绪论 1
1.1 大肠杆菌乳糖操纵子的基本概况 1
1.2 利用Red重组系统和Xer重组系统进行基因敲除 2
1.2.1 Red同源重组的作用机理和研究进展 2
1.2.2 Red同源重组的技术方法及技术难点 3
1.2.3 Xer重组系统的应用 4
1.3 本课题的研究思路和主要研究内容 4
2 材料与方法 7
2.1 实验材料 7
2.1.2 主要试剂和酶类 7
2.1.2 大肠杆菌基因删除试剂盒 8
2.1.3 菌株与质粒 8
2.1.4 引物 8
2.2 实验仪器和设备 9
2.3 培养基和试剂的制备 9
2.4 实验方法 10
2.4.1 PCR扩增lacY片段