CHARACTERIZATION OF M AERUGINOSA PLASMIDS FOR USE AS CLONING VECTORS

用作克隆载体的铜绿质粒的表征

• 批准号: 6240191
• 负责人: SHIRLEY RAPS
• 金额: $ 2.6万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240191
• 关键词: bacteria; bacterial toxins; cyclic peptides; gene expression; nucleic acid sequence; plasmids; protein structure function; restriction mapping; site directed mutagenesis; stoichiometry; transfection /expression vector

In order to understand the regulatory mechanisms involved in the coordinate expression of the PBS subunits, a search for a means of introducing exogenous DNA into Microcystic was initiated. The primary goal of this project is to develop a genetic system for studying this ecologically important cyanobacterium. We recently isolated an 8kb plasmid, named pMa025, from M.aeruginosa UV025. A hybrid plasmid was constructed by ligating pMa025 into pUC19. The resultant recombinant plasmid, pMaUC, is being maintained in E. coli XLI Blue. Our objective is to use this potential shuttle vector to introduce altered genes in Microcystic. The work to be carried out during this project includes the following. 1) Restriction mapping of pMa025 to enable us to a) delete segments to determine the approximate location of replicon sequences to ensure a viable plasmid and b) determine insertion sites for constructing recombinants. 2) Complete sequencing of the plasmid to ascertain if any information, such as open reading frames and possible procaryotic consensus promoter sequence, are present on the plasmid. This should provide insight regarding plasmid function(s). 3) Construction of a shuttle vector using the hybrid plasmid pMaUC. The appearance of ampicillin resistance provides evidence that pMaUC can transform M. aeruginosa. The vector will be streamlined to induce only pUC19, the genes involved in the replication and maintenance of pMa025 and a kanamycin resistance cassette. The resulting shuttle vector will be tested for its ability to introduce normal or altered genes into Microcystis and into E. coli. 4) Introduction of modified genes (after deletion or inactivation of endogenous genes) into Microcystic using the shuttle vector. Isolation of PBS subunit genes will be undertaken and methods will be developed for successful transformations using these or modified genes. 5) Characterization of pMa025 in association with its host. Very little is presently known regarding how cyano bacterial plasmids are maintained in vivo. In addition to identifying the replicon region, studies of the in vivo properties of pMa025 will be undertaken. These will include examining the mechanisms of plasmid partitioning and stability, quantitating copy number and assaying for variations in copy number with growth phase. 6) The possible role of plasmids in Microcystic toxin production will be evaluated.

为了了解涉及的监管机制， 协调PBS亚基的表达，寻找一种方法， 开始将外源DNA导入微囊藻。 主 该项目的目标是开发一个遗传系统来研究这一点。 生态上重要的蓝细菌。 我们最近分离出了一个8kb的 质粒，命名为pMa025，来自铜绿微囊藻UV025。 一个杂交质粒， 通过将pMa025连接到pUC 19中构建。 所得重组体 质粒pMaUC在E. coli XLI Blue。 我们的目标 是利用这种潜在的穿梭载体将改变的基因导入 微囊的。 本项目期间将开展的工作包括： 以下. 1)pMa025的限制性作图，使我们能够a）删除片段， 确定复制子序列的大致位置以确保 活质粒和B）确定用于构建插入位点 重组体 2)完成质粒测序以确定是否有任何信息， 例如开放阅读框和可能原核共有启动子 序列，存在于质粒上。 这应该能让我们 关于质粒功能。 3)使用杂合质粒pMaUC构建穿梭载体。 的 氨苄青霉素耐药性的出现提供了pMaUC可以 变换M.铜绿。 载体将被流线型化， pUC 19，参与pMa025复制和维持的基因 和卡那霉素抗性盒。 产生的穿梭载体将 测试其将正常或改变的基因导入 微囊藻和E.杆菌 4)引入修饰的基因（在缺失或失活修饰的基因之后）。 内源基因）转化微囊藻。 隔离 将进行PBS亚基基因的研究，并将开发用于 使用这些或修饰的基因的成功转化。 5)与其宿主相关的pMa025的表征。 很少 目前已知氰基细菌质粒如何维持 in vivo. 除了鉴定复制子区域之外， 将研究pMa025的体内性质。 这些将包括 研究质粒分配和稳定性的机制， 定量拷贝数并测定拷贝数的变化， 生长阶段 6)质粒在微囊藻毒素产生中的可能作用将是 评估。