DEVELOPMENT OF A GENETIC SYSTEM FOR BORRELIA BURGDORFERI

描述(改编自申请者的摘要)：最常见的莱姆病 在美国常见的节肢动物传播的疾病，是由感染引起的 与伯氏疏螺旋体有关。缺乏一套系统来 通过基因操作伯氏杆菌阻碍了对其研究的尝试 分子遗传学水平上的生物学和发病机制。几个组件 需要创建一种有用的遗传系统来分析 个体基因。这些包括可选择的标记、稳定的质粒载体、 和一种转化的方法。该项目建议开发一种基因 伯氏杆菌的系统，最初基于电穿孔和电阻 抗生素香豆素A1。此标记将用于停用 基因通过插入和选择来维持广泛的寄主范围 载体和重组天然质粒。此外，还可以选择其他 遗传标记将被筛选用于疏螺旋体系统。 建立这样的系统将有助于识别基因缺陷。 通过互补性突变菌株，将加快对质粒的研究 复制。几种不同抗生素载体的构建 抗性标记将允许基因在体外失活， 体内野生型基因座的替换和基因的互补 叛逃。这些基因技术将是系统化的 解剖生长和致病所需的基因。

DESCRIPTION (Adapted from the applicant's abstract): Lyme disease, the most common arthropod-borne disease in the United States, is caused by infection with the spirochete Borrelia burgdorferi. The lack of a system to genetically manipulate B. burgdorferi has hindered attempts to study its biology and pathogenesis on a molecular genetic level. Several components are required to create a useful genetic system for assaying the effect of individual genes. These include selectable markers, stable plasmid vectors, and a method of transformation. This project proposes to develop a genetic system for B. burgdorferi, initially based on electroporation and resistance to the antibiotic coumermycin A1. This marker will be used to inactivate genes by insertion and to select for the maintenance of broad host range vectors and recombinant natural plasmids. In addition, other selectable genetic markers will be screened for use in the borrelia system. Establishment of such a system will facilitate identifying gene defects in mutant strains by complementation and will expedite the study of plasmid replication. Construction of several vectors with different antibiotic resistance markers will permit the inactivation of genes in vitro, replacement of the wild type locus in vivo, and complementation of the defect. These genetic techniques will be the first steps in systematically dissecting the genes that are required for growth and pathogenesis.