芽孢杆菌表达系统的筛选和改造
毕业论文关键词：芽孢杆菌；表达系统；无痕敲除；筛选
The screening and modification of Bacillus expression system
Abstract：Bacillus is a gram-positive bacterium, it can secrete large amounts of protein to the outside of the cell, and its cell wall in the process won't produce endotoxin Most bacillus no pathogenicity for human, and its cell wall components mainly, peptidoglycan and teichoic acid in the process of secretory protein does not produce such as polysaccharide, difficult to remove material Therefore, bacillus expression system is a kind of important heterologous gene expression system in the study of this topic, soil milk samples collected from more than 10 provinces and cities nationwide, 6 strains of bacillus licheniformis appraisal separation Hope to be able to this strain as the carrier, construct efficient expression of exogenous gene expression system In addition, in order to improve the extracellular protease in bacillus subtilis expression efficiency, this topic is based on bacillus subtilis 168, adopt the method of non-trace knockout, builds a plant eight major extracellular protease gene nprE、aprE、epr、bpr、mpr、nprB、vpr、wprA strains of defects.
Key words: Bacillus; expression system; non-trace knock out.
目 录
摘要.1
关键词.1
Abstract.1
Key words.1
引言 3
1 材料与方法 4
1.1 材料及其培养 4
1.1.1 液体培养 4
1.1.2 固体培养 5
1.1.3 脱脂牛奶培养基 5
1.1.4 菌种保藏 5
1.2 地衣芽孢杆菌的分离筛选 5
1.2.1 土壤样品采集 5
1.2.2 土壤样品处理 5
1.2.3 土壤微生物分离纯化 5
1.2.4 土壤微生物鉴定 5
1.3 胞外蛋白酶基因的敲除 6
1.3.1 引物设计 6
1.3.2 同源臂的获得 7
1.3.3 同源臂的融合 7
1.3.4 融合片段转化 8
1.3.5 感受态的制作 9
1.3.5.1 感受态细胞的培养与转化 9
1.3.5.2 培养基配方 9
1.4 生长曲线的测定 9
1.5 无痕敲除方法技术路线 9
2 结果与分析 11
2.1 土壤样品分离菌株鉴定 11
2.2 nprE基因敲除鉴定 13
2.3 aprE基因敲除鉴定 14
2.4 epr基因敲除鉴定 15
2.5 bpr基因敲除鉴定 16
2.6 mpr基因敲除鉴定 17
2.7 nprB基因敲除鉴定 18
2.8 vpr基因敲除鉴定 19
2.9 wprA基因敲除鉴定 20