Optimization of Mariner Transposon for Bacillus

描述（由申请人提供）：转座因子是细菌遗传分析中最有用的工具之一。转座子不仅可以破坏操纵子或用选择标记标记感兴趣的等位基因，而且这些元件的携带复制起点或报告基因的专门版本可以用于直接克隆或分析它们破坏的操纵子的表达。包括炭疽杆菌在内的革兰氏阳性菌的研究得益于在B中开发的转座子系统（Tn917）。枯草杆菌。尽管Tn917系统已成功用于许多实例，但它并非没有局限性。最值得注意的是，99%的所有Tn917易位在B中。枯草杆菌靶向B上的“热点”。枯草染色体最近，真核转座因子Himar 1已被修饰用于许多细菌系统，其中随机转座发生成二核苷酸序列（TA）。本申请寻求开发用于芽孢杆菌属物种的Himar 1。在芽孢杆菌中可选择的抗生素抗性盒将被转座子的反向末端重复序列（ITR）包围，并置于具有温度敏感性芽孢杆菌复制起点的质粒上。质粒还将包括具有适合于芽孢杆菌属的表达元件的Himar 1转座酶基因的高活性变体。该系统将在遗传上更易处理的B中进行评估。枯草芽孢杆菌，并且一旦优化，就用另外的元素（例如，报告基因系统，用于克隆的复制起点）并在B中测试有效性。炭疽病在开发过程中，Himar 1转座酶变体，在芽孢杆菌中过度活跃，将使用为大肠杆菌开发的乳头状试验的修改和芽孢杆菌特有的孢子形成选择方案来寻求。在这项研究中创建的转座子应该在芽孢杆菌属物种和革兰氏阳性细菌中使用。此外，本研究中分离的高度活跃的Himar 1转座酶变体可以并入现有的Himar 1系统以提高其效率，并提供对转座酶活性或稳定性重要的元件的见解。

DESCRIPTION (provided by applicant): Transposable elements are among the most useful tools available for genetic analysis in bacteria. Not only can transposons disrupt operons or tag interesting alleles with selectable markers, but specialized versions of these elements, carrying origins of replication or reporter genes, can be used to directly clone or analyze the expression of the operons that they disrupt. Studies of Gram-positive bacteria, including Bacillus anthracis have benefited from a transposon system (Tn917) developed in B. subtilis. Although the Tn917 system has been successfully used in a number of instances, it is not without its limitations. Most notably, 99% of all Tn917 transpositions in B. subtilis target "hot spots" on the B. subtilis chromosome. Recently, the eukaryotic transposable element Himar1 has been modified for use in a number of bacterial systems, where random transposition occurs into a dinucleotide sequence (TA). The current application seeks to develop Himar1 for use in Bacillus species. Antibiotic resistance cassettes, selectable in Bacillus will be bracketed by the transposon's inverted terminal repeats (ITR) and placed on a plasmid with a temperature-sensitive Bacillus origin of replication. The plasmid will also include a hyperactive variant of the Himar1 transposase gene with expression elements appropriate for Bacillus. The system will be evaluated in the more genetically tractable B. subtilis, and once optimized, embellished with additional elements (e.g., a reporter gene system, origin of replication for cloning) and tested for effectiveness in B. anthracis. During development, Himar1 transposase variants, hyperactive in Bacillus will be sought using a modification of a papillation assay developed for use in Escherichia coli and a sporulation selection protocol unique to Bacillus. The transposons created in this study should be of use both in Bacillus species and Gram-positive bacteria in general. Additionally, the hyperactive Himar1 transposase variants isolated in this study could be incorporated into existing Himar1 systems to enhance their efficiency, and provide insights into elements of the transposase that are important for its activity or stability.