CHARACTERIZATION OF M AERUGINOSA PLASMIDS FOR USE AS CLONING VECTORS

为了了解涉及的监管机制 PBS亚基的坐标表达，寻找一种方法 开始将外源DNA导入微囊中。初级阶段 这个项目的目标是开发一个用于研究这一现象的遗传系统。 生态上重要的蓝藻。我们最近分离出了一个8KB的 质粒pMa025，产自铜绿假单胞菌UV025。构建了一个杂交质粒。 将pMa025连接到pUC19构建而成。由此产生的重组 质粒pMaUC被保存在大肠杆菌Xli Blue中。我们的目标 是使用这个潜在的穿梭载体将改变的基因引入到 微囊症。在这项工程进行期间进行的工作包括 跟在后面。 1)pMa025的限制映射，使我们能够a)删除片段以 确定复制子序列的大致位置以确保 活质粒和b)确定构建的插入位置 重组体。 2)完成对该质粒的测序以确定是否有任何信息， 例如开放阅读框架和可能原核共识启动子 序列，都存在于该质粒上。这应该会提供洞察力 关于质粒功能(S)。 3)利用pMaUC杂合载体构建穿梭载体。这个 氨苄西林耐药的出现为pMaUC可以 转化铜绿假单胞菌。该向量将被简化为仅诱导 PUC19，参与pMa025复制和维持的基因 还有一盒卡那霉素抗性录像带。由此产生的穿梭向量将 被测试是否有能力将正常或改变的基因引入 微囊藻进入大肠杆菌。 4)引入修饰基因(在缺失或失活之后 内源基因)利用穿梭载体导入微囊。隔离 将进行PBS亚单位基因的研究，并将制定方法 使用这些或修饰的基因成功转化。 5)pMa025与其宿主相关的特性。很少 目前已知氰基细菌质粒是如何维持的 在活体内。除了确定复制子区域外，对复制子区域的研究 将进行pMa025的体内性质研究。这些措施包括 检查质粒分割和稳定性的机制， 定量拷贝数并分析拷贝数的变异 生长期。 6)质粒在微囊藻毒素生产中的可能作用 已评估。

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.