pPopctrl质粒中Tn5转座子的插入对lux box启动的影响
关键词：细菌自杀，群体感应，突变，转座子
The impact of the insertion of Tn5 transposon to the start of lux box in the pPopctrl
Abstract：The bacteria with suicide gene circuit based on Quorum-Sensing will conduct killing themselves in a controllable pattern upon certain cell density. In early period of this research, we observed the growth and suicidal behavior of the E. coli. Top10F’ with such gene circuit, screened the mutants and determined their mutated loci in the media of different IPTG inducer concentration, The results showed that, with higher IPTG concentration, the more wild type bacteria were killed; as well the mutants emerged earlier and spread over the population more quickly. The sequence of plasmids in those mutants revealed that a transposon inserted into the luxR gene and therefore disrupted Quorum-Sensing of these individuals. Furthermore, it was also testified that the insertion sequence of the plasmid can solely result in the mutants escaping from suicide. In the late period of the experiment, there is a new plan that a new plasmid called LuxR-GFPuv2 will be constructed which will be used to find out the effect of the transposon to the expression of the luxR. Although we did not achieve these goals in the end because of time issues, it still provides us a new idea on the next step of the experiment.
Keywords：bacterial suicide，Quorum Sensing, mutation, transposon
目 录
1. 前言 1
1.1. 细菌的群体感应 1
1.2. 自杀基因回路设计 1
1.3. LuxR蛋白基本结构特点 3
1.4. 抗自杀突变种群 4
1.5. 本研究主要内容及其意义 4
2. 材料与方法 5
2.1. 材料与仪器设备 5
2.1.1. 菌种 5
2.1.2. 实验材料 6
2.1.3. 主要仪器和设备 7
2.1.4. 培养基及试剂的制备 8
2.2. 实验方法 9
2.2.1. 细菌扩大培养 9
2.2.2. 菌株筛选 9
2.2.3. 突变菌株的质粒提取和测序 9
2.2.4. 突变质粒转化不含任何质粒的top10F’菌株后的生长曲线测定 9
2.2.5. 扩菌保种 10
2.2.6. 构建质粒LuxR-GFPuv2 10
3. 实验结果与分析 14
3.1. 突变菌株的筛选和自杀过程中突变种群的蔓延 14
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