Non-antiobiotic resistance markers for bacteria

描述（由申请人提供）：许多NIAID A类、B类和C类优先病原体（也被列为选择性病原体细菌）的基因组序列的完成，现在极大地促进了旨在了解这些细菌的生物学和发病机制的遗传实验的设计和执行。然而，这些努力由于以下事实而变得非常复杂：当今可用的大多数遗传工具依赖于抗生素选择标记的使用，其中许多抗生素选择标记被NIH重组DNA研究指南禁止用于这些细菌，因为它们可能损害相应药物的治疗用途。我们假设，属于杀生物剂，除草剂和重金属家族的非抗生素抗性标记可以被工程化，以增强选择代理列表上的细菌可用的遗传工具的库。为了检验这一假设，将追求两个具体目标。在目标1中，我们将修改分别编码三氯生（杀生物剂）、双丙氨磷和氯嘧磺隆乙酯（除草剂）以及亚碲酸盐（重金属）抗性的fabL、bar和ILV以及kilAtelAB基因。通过将各个基因置于修饰的大肠杆菌lac操纵子启动子的控制下来构建表达盒。酵母Flp重组酶靶（FRT）位点将提供用于体内切除并因此回收选择性标记的选择。在目标2中，将以鼻疽伯克霍尔德氏菌B为例，测试新构建的标记在优先病原体研究中的适用性。类鼻疽菌、土勒弗朗西斯菌和鼠疫耶尔森菌。这将通过构建和测试具有Flp重组酶介导的标记切除选项的多拷贝复制型和单拷贝基因整合载体来完成。最后，我们期望开发出一组通用的遗传工具，其中包含用于选择药剂B的不同非抗生素抗性标记。鼻疽B. mallei，B. pseudomallei、F. tularensis和Y.鼠疫以及其他细菌病原体，对于这些细菌病原体，人们在使用抗生素选择标记进行遗传操作方面面临类似的限制。

DESCRIPTION (provided by applicant): The completion of the genome sequences for many NIAID category A, B and C priority pathogens, which are also listed as select agent bacteria, now greatly facilitates the design and execution of genetic experiments aimed at understanding the biology and pathogenesis of these bacteria. However, these endeavors are greatly complicated by the fact that most genetic tools available today rely on the use of antibiotic selection markers, many of which are prohibited for use in these bacteria by NIH Guidelines for Recombinant DNA Research because they potentially compromise the therapeutic use of the respective drugs. We hypothesize that non-antibiotic resistance markers belonging to the biocide, herbicide and heavy metal families can be engineered to enhance the repertoire of genetic tools available for bacteria on the select agent list. To test this hypothesis, two specific aims will be pursued. In aim 1, we will modify the fabL, bar and ILV, and kilAtelAB genes, which encode triclosan (biocide), bialaphos and chlorimuron ethyl (herbicide), and tellurite (heavy metal) resistance, respectively. Expression cassettes will be constructed by placing the respective genes under the control of modified Escherichia coli lac operon promoters. Yeast Flp recombinase target (FRT) sites will provide an option for in vivo excision and therefore recycling of the selectable markers. In aim 2, the applicability of the newly constructed markers for priority pathogen research will be tested by using the examples of Burkholderia mallei, B. pseudomallei, Francisella tulerensis and Yersinia pestis. This will be done by construction and testing of multi-copy replicative and single-copy gene integration vectors with the option of Flp recombinase-mediated marker excision. In the end, we expect to have developed a panel of versatile genetic tools containing different non-antibiotic resistance markers for use in the select agents B. mallei, B. pseudomallei, F. tularensis and Y. pestis, as well as other bacterial pathogens for which one faces similar restrictions regarding use of antibiotic selection markers for genetic manipulation.