Advanced Genetic Tools for Studying Chlamydia

研究衣原体的先进遗传工具

• 批准号: 10593742
• 负责人: Anthony T Maurelli
• 金额: $ 22.46万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10593742
• 关键词: Animals; Antibiotics; Applications Grants; Arabinose; Bacteria; Bacterial Physiology; Bacterial Sexually Transmitted Diseases; Blindness; Chlamydia; Chlamydia trachomatis; Chromosomal Insertion; Chromosomes; Communities; Complement; Development; Disease; Effectiveness; Escherichia coli; Frequencies; Future; Gene Expression; Genes; Genetic; Genetic Complementation Test; Genetic Enhancement; Genetic study; Genome; Human; Individual; Lac Operon; Maintenance; Measures; Operon; Paper; Phenotype; Plasmids; Population; Property; Publishing; Reporter Genes; Reporting; Repression; Research; Research Project Grants; Seminal; Site; System; Testing; Transgenes; Transposase; United States; Validation; complement system; design; expression cloning; flexibility; gene cloning; gene complementation; genetic analysis; genetic manipulation; genital infection; improved; innovation; interest; mutant; overexpression; pathogenic bacteria; prevent; promoter; sound; tool

PROJECT SUMMARY/ABSTRACT Chlamydiae are obligate intracellular bacterial pathogens that cause disease in human and animal populations Chlamydia trachomatis is the major cause of both bacterial sexually transmitted disease and infectious blindness in the world. Despite great strides over the past decade, tools for genetic study and manipulation of Chlamydia spp. remain severely limited. This proposal will develop new and powerful genetic tools including tightly negatively regulated promoter expression systems, a Tn7-based single chromosomal insertion transgene system, and application of the latter to build new, smaller shuttle plasmids for transformation. Specific Aim 1 – Development of a tightly negatively regulated inducible promoter for Chlamydia. This aim will provide one of the key missing tools in the repertoire of genetic tools for Chlamydia: a tightly negatively regulated, inducible, and titratable promoter for performing expression / overexpression studies. In bacterial physiology studies, it is important to be able to express and overexpress cloned genes in a mutant or wild type genetic background. One also must be able to tightly repress the cloned gene to avoid undesirable phenotypes associated with leaky expression of the cloned gene. The two subaims will design and test inducible promoter systems derived from two well-established Escherichia coli systems: the lac operon and the arabinose operon. Both systems are tightly repressed in the absence of inducer and rapidly upregulated when inducer is present. Our rigorous validation plan will use three reporter genes to measure promoter activity in the presence and absence of inducer. The lac operon promoters will allow us to develop a range of promoters of varying induction strength and induction ratios to provide flexibility for future genetic studies. Specific Aim 2 – Development of a chromosomal Tn7 transgene insertion system for single copy gene complementation studies in Chlamydia. Shuttle plasmids used in Chlamydia complementation studies are based on the native Chlamydia plasmid, which is present at 7.6 copies per genome. The major shortcoming of expressing a cloned gene from a multi-copy plasmid is that even low copies of the plasmid may lead to non-physiological levels of gene expression and aberrant phenotypes that complicate interpretation of the complementation results. The Tn7 transgene system provides high frequency, site- specific, single copy chromosomal insertion of any gene with the additional advantage of removing the need for antibiotic selection for transgene maintenance. We will adapt the Tn7 transgene system to Chlamydia. Our proposal includes a rigorous strategy to test the transgene system and plans to optimize the system by selection for transposase mutants that more efficiently recognize the chlamydial Tn7 attachment site. We will apply the Tn7 transgene system to build and validate new, smaller shuttle plasmids, which should improve transformation efficiencies. We will make all tools developed in this proposal available to the research community. Thus, successful completion of these aims will enhance genetic studies in Chlamydia.

项目概要/摘要 衣原体是引起人类和动物群体疾病的专性细胞内细菌病原体 沙眼衣原体是性传播疾病和传染病的主要原因 尽管过去十年取得了巨大进步，但基因研究和操纵的工具仍然存在。 该提案将开发新的强大的遗传工具，包括衣原体属。 严格负调控启动子表达系统，基于 Tn7 的单染色体插入 转基因系统，并应用后者构建新的、更小的穿梭质粒进行转化。 具体目标 1 – 开发衣原体严格负调控诱导型启动子。 这一目标将为衣原体遗传工具库中提供一个关键的缺失工具：一个紧密的 用于进行表达/过表达研究的负调控、诱导型和可滴定启动子。 在细菌生理学研究中，能够在突变体或突变体中表达和过度表达克隆基因非常重要 野生型遗传背景还必须能够严格抑制克隆基因以避免不良后果。 与克隆基因的渗漏表达相关的表型将设计和测试。 诱导型启动子系统源自两个成熟的大肠杆菌系统：lac操纵子和 阿拉伯糖操纵子在没有诱导物的情况下受到严格抑制，并且在没有诱导物的情况下迅速上调。 我们严格的验证计划将使用三个报告基因来测量启动子活性。 在存在和不存在诱导物的情况下， lac 操纵子启动子将使我们能够开发一系列的 不同诱导强度和诱导比率的启动子，为未来的遗传研究提供灵活性。 具体目标 2 – 开发单拷贝染色体 Tn7 转基因插入系统 用于衣原体互补的穿梭质粒的基因互补研究。 研究基于天然衣原体质粒，每个基因组有 7.6 个拷贝。 从多拷贝质粒表达克隆基因的缺点是，即使是低拷贝的质粒也可能 导致基因表达的非生理水平和异常表型，使情况复杂化 Tn7 转基因系统提供高频率、位点解释。 任何基因的特异性、单拷贝染色体插入，具有去除 需要选择抗生素来维持转基因。我们将使 Tn7 转基因系统适应衣原体。 我们的提案包括测试转基因系统的严格策略，并计划通过以下方式优化系统： 选择更有效地识别衣原体 Tn7 附着位点的转座酶突变体。 将应用 Tn7 转基因系统来构建和验证新的、更小的穿梭质粒，这应该 我们将把本提案中开发的所有工具提供给研究。 因此，成功完成这些目标将加强衣原体的遗传学研究。