食甲基丁酸杆菌高效电转化方法的建立

食甲基丁酸杆菌高效电转化方法的建立(论文10000字)
摘要
食甲基丁酸杆菌（Butyribacterium methylotrophicum）可以同时利用多种不同的C1原料进行发酵，如CO2、CO、甲醇等，同其它菌株相对，其对甲醇的利用速率表现出明显的优势，因此开发食甲基丁酸杆菌作为甲醇利用的模式宿主，对促进甲醇的生物转化具有重要意义。然而当前研究中该菌株遗传操作工具的缺乏，限制了其的发展和应用。高效的转化策略是进行分子操作的基础，本文为建立食甲基丁酸杆菌高效的转化体系，考察了不同甲基化酶修饰对质粒转化效率的影响，同时对比分析了不同复制子的质粒对菌株的转化效率，确定了可在食甲基丁酸杆菌中稳定存在的复制子，此外为进一步提高转化的效率，分别从感受态细胞的制备过程（细胞壁弱化剂、细胞膜增溶剂）、电转参数（电压、电阻）、孵育过程（时间）三个方面进行系统优化，最终获得了高效的电转策略，质粒转化效率可达3170，较未优化前提高了约10倍。

关键词：甲醇 甲基化 复制子 转化

Establishment of high efficient electroporation method for Butyribacterium methylotrophicum
Abstract
Buttyribacterium methylotrophicum can simultaneously utilize a variety of C1 raw materials, such as CO2, CO, methanol, etc.Compared with other strains, which shows a significant optimization of the utilization rate of methanol, so the development of methylbutyric acid As a model host for methanol utilization, Bacillus is important for promoting the biotransformation of methanol. However, the lack of genetic manipulation tools for this strain in the current study limits its development and application. Efficient transformation strategy is the basis of molecular manipulation. In order to establish an efficient transformation system of M. acidobacillus, the effects of different methylation enzyme modifications on plasmid transformation efficiency were investigated, and the plasmid pairs of different replicons were compared and analyzed. The transformation efficiency of the strain determines the replicon that can be stably present in M. acidobacillus, and further increases the efficiency of transformation from the preparation process of competent cells (cell wall weakening agent, cell membrane solubilizing agent), electrical parameters System optimization was carried out in three aspects (voltage, resistance) and incubation process (time). Finally, an efficient electrotransformation strategy was obtained. The plasmid conversion efficiency reached 3170, which was about 10 times higher than before optimization.
Key words: Methanol; methylation; replicon; transformation

目录
摘要    I
Abstract    II
第一章 文献综述    1
1.1甲醇的概要    1
1.1.1甲醇    1
1.1.2甲醇的市场    1
1.2食甲基丁酸杆菌性质概要    2
1.2.1食甲基丁酸杆菌    2
1.2.2食甲基丁酸杆菌发酵甲醇    2
1.2.3建立食甲基丁酸杆菌合适遗传操作体系的必要性    3
1.3 本论文的研究内容以及拟采用的手段    3
1.4本论文的研究意义    3
第二章 实验部分    5
2.1仪器与试剂    5
2.1.1仪器    5
2.1.2试剂    6
2.1.3培养基    6
2.2质粒构建    8
2.2.1基因克隆    8
2.2.2 酶切、连接和转化    9
2.2.3菌落PCR验证    10
第三章 实验结果    12
3.1食甲基丁酸杆菌感受态细胞的制备及质粒转化的探索    12
3.2 食甲基丁酸杆菌的固体培养基的筛选    12
3.3甲基化修饰系统及复制子对转化效率的影响分析    13
3.4食甲基丁酸杆菌感受态细胞制备的优化    15
3.4.1添加细胞壁弱化剂    15
3.4.2添加细胞膜弱化剂    16
3.4.3优化复苏时间    17
3.4.4优化质粒DNA添加量    18
3.4.5添加细胞洗涤液    19
3.5电转过程中的参数优化    20
3.5.1优化电压    20
3.5.2优化电阻    21
3.6优化后的转化策略    22
3.7讨论    23
第四章 总结    24
参考文献    25
致谢    28