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摘要：细菌的自杀行为（野生型 ）是一种自然现象。对这一现象的产生和文持及其对细菌群体进化的影响的研究在理论和实践上都具有重要意义。在细菌自杀过程中，不可避免的会产生变异现象（突变型）。突变菌株与野生菌株的的差异在于pPopctrl质粒中有Tn5转座子的插入。有研究证明仅仅依靠质粒上的转座子的插入，就足以对抗IPTG诱导的自杀行为，但pPopctrl质粒中Tn5转座子的插入对AHL信号分子的生成是否产生影响尚未明了。本课题论文从先前得到的125菌株中提取质粒，转化到107菌株中，得到新型菌株，命名为107’菌株（top10F’为宿主，仅含pLuxCcdB3(来自107菌株)及pLuxR2(来自125菌株)）。比较107’菌株在野生型、突变型各自的TBK培养基（均含IPTG,以不含IPTG作对照）上清液中的生长规律差异发现，107’菌株在野生菌株的不同TBK上清液中生长时有明显差异，发生自杀现象；而在突变菌株的不同TBK上清液中生长时无明显差异，未发生自杀现象。得出结论pPopctrl质粒中Tn5转座子的插入对AHL信号分子的生成有影响。6504
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关键词： 细菌自杀；pPopctrl质粒；Tn5转座子；IPTG；AHL
Effect of insertion of transposon Tn5 in plasmid pPopctrl on the formation of AHL signaling molecules
Abstract: Suicide behavior （wild-type）of bacterium is a natural phenomenon. Study on gain and maintenance of suicide phenomenon and effect of this phenomenon on the evolution of bacterial population is important in theory and practice. In the process of bacterium suicide, mutation is inevitable（mutant）. Difference between wild-type and mutant is the insertion of transposon Tn5 on plasmid pPopctrl. It is proved that it is enough to fight IPTG induced suicide by inserting transposon to plasmid. Whether insertion of transposon Tn5 influences generation of signaling molecule AHL has not yet known. We have got two strains named 107 strain and 125 strain in previous work. In this study, plasmid DNA was extracted from 125 strain and was transformed into 107 strain. Then we got a new strain and named it as 107’ strain (with 'top10F' as the host, plasmid containing only pLuxCcdB3(from 107 strain) and pLuxR2(from 125 strain). We compared growth patterns of 107' in the supernatant of wild-type strain and mutant strain of their TBK medium (respectively, containing IPTG, no IPTG as control) and found that there were significant differences :107’ strain in different TBK supernatants of wild-type strains occurred suicide behavior while 107’ strain in different TBK supernatant of mutants grown well.We finally inferred that the insertion of transposon Tn5 has influence on generation of signaling molecule AHL.
Key Words: bacterium suicide; pPopctrl plasmid;Tn5 transposons ; IPTG; AHL
目录
1 绪论 1
1.1 细菌的自杀行为 1
1.2群体感应现象 1
1.3 AHL信号分子概述 1
1.4 细菌自杀基因回路 2
1.5 细菌的突变 3
1.6本课题研究的内容和意义 4
2.1使用的材料试剂、仪器设备 5
2.2 采取的方法 6
2.2.1 培养基配制 6
2.2.2 基本操作 7
2.2.3 质粒的抽提、检测、转化 7
2.2.4 CFU数据测定 10
2.2.5酶标仪法测定生长曲线 10
3实验过程及结果分析 12
3.1比较野生型、突变型及新菌种107’在新鲜TBK培养中的生长差异 12
3.1.1转化新菌种及菌种的确定 12
3.1.2比较野生型、突变型及新菌种在新鲜TBK培养基中的生长特征 13
3.2 CFU法比较107’菌种在野生型及突变型TBK上清液中的生长差异 14