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摘要：自杀型质粒pPopctrl转化宿主细胞后赋予了宿主在生长到一定密度时启动自杀的特性。在本课题前期研究阶段，为了使基因回路更具鲁棒性，在转化后，以不同浓度的IPTG为诱导剂，观察宿主菌的表型特征以及在自杀过程中突变菌产生的规律，并通过基因测序确定变异位点。结果表明：IPTG浓度与细菌自杀率呈正相关，自杀强度愈大，突变菌株出现得越早，蔓延的速度也越快。基因测序结果表明，在基因回路质粒上，luxR基因中间有转座子插入，从而破坏了群体感应系统。实验也表明：仅需该突变，就足以使宿主菌逃避自杀。在本课题研究的中后期，计划在前期研究的基础上，通过人工构建新质粒LuxR-GFPuv2来进一步探究该类型转座子的插入对luxR基因表达的影响，以及生成的变异R蛋白对lux box启动子的影响。虽然由于时间原因最终没得到理想的结果，但还是为将来下一步构建更具鲁棒性的基因回路提供了思路。4114
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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