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摘要：在标准PCR反应体系中，以芽孢杆菌SL16的基因组为模板，通过改变镁离子的浓度和加入锰离子即连续易错PCR的实验方法获得发生多个点突变的目的基因。把从大肠杆菌DH5α中提取的质粒pET-32a(+)和通过点突变获得的纤维素酶目的基因用HindⅢ和BamHⅠ分别进行双酶切后再纯化回收，用T4-DNA连接酶将其连接起来构建pET-32a-egls重组质粒。将其导入大肠杆菌DH5α感受态细胞中进行转化，再将转化子接种在含氨苄青霉素的LB固体培养基上进行抗性筛选。结果表明易错PCR体系镁离子浓度为7mM，锰离子浓度为0.5mM时突变效果最好，获得的点突变目的基因电泳结果条带最亮，转化过程中接种有重组质粒表达载体的含氨苄青霉素的LB固体培养基出现许多白色菌落，表明转化成功。49879
毕业论文关键词：点突变；易错PCR；纤维素酶；突变体库
Construction of cellulase mutagenesis library by site mutation
Abstract: In the standard PCR reaction, the genome of Bacillus sp. SL16 was used as template. The target genes of contain site mutations by changing the concentration of magnesium ion and adding manganese ion were obtained through error-prone polymerase chain reaction (EP-PCR). The plasmid that extracted from E. coli DH5α and the target gene which obtained by EP-PCR were double digested with HindⅢand BamHⅠ. After the purification of the enzyme digested PCR product, Connect it with the T4 DNA ligase - build pET - 32 a - egls recombinant plasmid. Then, they were transformed into the competent cells of E. coli DH5α. Positive transformants were screened on solid LB agar containing 100ug/ml ampicillin. The results shown that in the standard PCR reaction, efficiency of site mutation is best when the concentration of magnesium ion was 7mM and the concentration of manganese ion was 0.5mM.
Key words: Site mutation; Error-prone PCR; Cellulase ; Mutagenesis library
目 录
摘要 1
Abstract 1
引言 2
1. 材料与方法 2
1.1 实验材料 2
1.1.1 实验试材 2
1.1.2 主要培养基 2
1.1.3 试剂 3
1.2 实验仪器与设备 4
1.3 实验方法 4
1.3.1 纤维素酶基因的获得 4
1.3.1.1 SL16基因组的提取 4
1.3.1.2引物的设计 5
1.3.1.3 PCR扩增 5
1.3.1.4 PCR扩增产物的纯化回收 5
1.3.2 易错PCR 6
1.3.2.1 易错PCR模板的制备 6
1.3.2.2 易错PCR条件的优化 6
1.3.2.3 易错PCR产物的纯化回收 7
1.3.3 质粒载体的制备 7
1.3.4 易错PCR产物与质粒的双酶切 7
1.3.4.1 易错PCR产物的双酶切 8
1.3.4.2 质粒的双酶切 8
1.3.5 双酶切产物的连接 8
1.3.6 连接产物的转化 9
1.3.6.1 菌体的培养 9
1.3.6.2感受态细胞的制备