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

描述(由申请人提供)： 如果基于非抗生素可选标记的不受监管和安全的遗传工具可用于基因操作，那么现在可用于具有重大生物恐怖风险的精选制剂的大量细菌基因组应该有助于各种生物防御研究。转座子、复制质粒、等位基因替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-宿主相互作用方面发挥了关键作用，这些因素已导致疫苗、治疗和诊断靶点。对于细菌选择剂来说，构建多个抗生素耐药菌株是危险的，而且在美国受到限制，这阻碍了对生物防御具有重要意义的研究，需要开发非抗生素可选标记。该项目的长期目标是开发完全基于非抗生素标记(S)的这些遗传工具，用于布鲁氏菌和伯克霍尔德氏菌物种基因组的标准重复操作，这些操作可能延伸到其他具有重大生物恐怖主义风险的选定制剂。根据我们的初步数据和正在进行的研究表明，独特的FRT序列可以帮助回收可选择的标记进行几乎无限的基因组操作，我们的工作假设和前提是，当任何给定的物种与FLP-FRT切除系统耦合时，对于任何给定的物种，只需要一个合适的非抗生素可选择标记。由于我们成功地利用独特的FRT序列操纵了各种细菌基因组，而没有基因组的不良删除或重排，我们将扩大FLP-FRT的使用，以开发布鲁氏菌和伯克霍尔德氏菌抗汞(MERR)的新型遗传工具，具体如下：i)我们将开发和测试布鲁氏菌(流产布鲁氏菌、山羊布鲁氏菌和猪布鲁氏菌)和伯克霍尔德氏菌(马利氏杆菌和伪马利氏杆菌)的新型配对供体和克隆菌株和穿梭载体。将使用的两个非抗生素可选标记是ASD(天冬氨酸-半乳糖醛脱氢酶)和MERR。Ii)我们将开发能够适应重复的等位基因替换和转座子突变的FRT-MERR-FRT盒。这些Merr盒的用途将在流产杆菌和马来杆菌的等位基因替换实验和假鼻疽杆菌的转座子突变实验中得到证明。

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.