Development of Genetic Systems Based on Non-antibiotic Selectable Markers for Bru

描述（由申请人提供）： 如果基于非抗生素选择标记的不受管制和安全的遗传工具可用于遗传操作，那么现在可用于选择具有重大生物恐怖风险的药剂的众多细菌基因组应有助于生物防御的各种研究。转座子、复制质粒、等位基因置换系统和接合供体菌株等工具在帮助研究人员研究分子遗传学、发病机制和细菌-宿主相互作用方面至关重要，这些工具导致了疫苗、治疗和诊断靶标的产生。对于细菌选择剂，构建多种抗生素抗性菌株是危险的，并且在美国受到限制，这阻碍了对生物防御重要的研究，并且需要开发非抗生素选择标记。该项目的长期目标是开发这些遗传工具，仅基于非抗生素标记，用于布鲁氏菌和伯克霍尔德氏菌属物种基因组的标准重复操作，这些操作可能扩展到具有重大生物恐怖主义风险的其他选定制剂。基于我们的初步数据和正在进行的研究，表明独特的FRT序列可以帮助回收选择性标记用于几乎无限的基因组操作，我们的工作假设和前提是，当它与Flp-FRT切除系统偶联时，对于任何给定的物种，只需要一个合适的非抗生素选择性标记。由于我们成功地使用独特的FRT序列操纵各种细菌基因组，而没有不希望的基因组缺失或重排，我们将扩展Flp-FRT的使用，以开发用于布鲁氏菌和伯克霍尔德氏菌属物种的抗汞（MerR）的新型遗传工具，如下：i）我们将开发和测试用于布鲁氏菌（B. abortus，B. melitensis和B. suis）和伯克霍尔德氏菌（B.鼻疽和假鼻疽）。将使用的两个非抗生素选择标记是Asd（谷氨酸-乙醛脱氢酶）和MerR。ii）我们将开发FRT-MerR-FRT盒，其适合于重复轮的等位基因置换和转座子诱变。这些MerR盒的有用性将在B的等位基因置换实验中得到证实。流产和B.对B进行鼻疽和转座子诱变。假鼻疽

DESCRIPTION (provided by applicant): The numerous bacterial genomes now available for select agents of significant bioterror risks should aide various studies for biodefense, if unregulated and safe genetic tools based on nonantibiotic selectable markers are available for genetic manipulations. Tools such as transposons, replicating plasmids, allelic-replacement systems, and conjugation donor strains have been critical in aiding investigators to study molecular genetics, pathogenesis, and bacteria-host interactions, which have led to vaccine, therapeutic, and diagnostic targets. For bacterial select agents, construction of multiple antibiotic resistant strains is dangerous and is restricted in the United States, which hampers studies important for biodefense and requires the need to develop non-antibiotic selectable markers. The long-term goal of this project is to develop these genetic tools, based solely on non-antibiotic marker(s), for standard repetitive manipulations of the genome of Brucella and Burkholderia species that may extend into other select agents of significant bioterrorism risks. Based on our preliminary data and ongoing research indicating that unique FRT sequences can help recycle selectable markers for almost unlimited genome manipulations, our working hypothesis and premise is that there is a requirement for only one suitable non-antibiotic selectable marker for any given species when it is coupled to the Flp-FRT excision system. Because of our success in manipulating various bacterial genomes using unique FRT sequences without undesirable deletions or rearrangements of the genome, we will extend the use of Flp-FRT to develop novel genetic tools with the mercury-resistant (MerR) for Brucella and Burkholderia species as follows: i) We will develop and test novel conjugal donor and cloning strains and shuttle-vectors for Brucella (B. abortus, B. melitensis, and B. suis) and Burkholderia (B. mallei and pseudomallei). The two non-antibiotic selectable markers that will be used are Asd (aspartate-seminaldehyde dehydrogenase) and MerR. ii) We will develop FRT-MerR-FRT cassettes that are amenable to repetitive rounds of allelic replacement and transposon mutagensis. The usefulness of these MerR-cassettes will be demonstrated in allelic-replacement experiments for B. abortus and B. mallei and transposon mutagenesis for B. pseudomallei.