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

基于 Bru 非抗生素选择标记的遗传系统的开发

基本信息

批准号：
7287451
负责人：
Tung T Hoang
金额：
$ 19.8万
依托单位：
UNIVERSITY OF HAWAII AT MANOA
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7287451
关键词：
Aleurites Antibiotic Resistance Antibiotics Aspartate Aspartate-Semialdehyde Dehydrogenase Bacteria Bacterial Genome Bioterrorism Brucella Brucella abortus Brucella melitensis Burkholderia Burkholderia mallei Burkholderia pseudomallei Categories Cells Cloning Cloning Vectors Complement Country Coupled DNA Sequence Rearrangement Data Development Diagnostic Engineering Escherichia coli Excision Facility Construction Funding Category Frequencies Funding Gene Expression Genes Genetic Genetic Recombination Genetic screening method Genome Genus Mycobacterium Goals Grant Human Infection Laboratories Libraries Literature Mercury Molecular Genetics Multi-Drug Resistance Mutagenesis Mutation National Institute of Allergy and Infectious Disease Operon Organism Oxidoreductase Pathogenesis Physiology Plasmids Positioning Attribute Principal Investigator Publishing Range Recycling Replication Origin Research Research Personnel Resistance Risk Security Shuttle Vectors Side Standards of Weights and Measures System Testing United States United States National Institutes of Health Vaccines Work Yeasts base biodefense concept experience gene cloning genetic manipulation killings mutant novel programs research study response success suicidal therapeutic vaccine tool vector

项目摘要

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.

描述（由申请人提供）：如果基于非抗生素选择标记的不受监管且安全的遗传工具可用于遗传操作，那么现在可用于具有重大生物恐怖风险的选定代理的众多细菌基因组应该有助于生物防御的各种研究。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对于帮助研究人员研究分子遗传学、发病机制和细菌-宿主相互作用至关重要，从而产生了疫苗、治疗和诊断靶点。对于细菌选择剂来说，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仅基于非抗生素标记的遗传工具，用于对布鲁氏菌和伯克霍尔德氏菌物种的基因组进行标准重复操作，这些操作可能会扩展到其他具有重大生物恐怖主义风险的选定病原体。根据我们的初步数据和正在进行的研究表明，独特的 FRT 序列可以帮助回收选择性标记以进行几乎无限的基因组操作，我们的工作假设和前提是，对于任何给定物种，当它需要时，只需要一种合适的非抗生素选择性标记。与 Flp-FRT 切除系统耦合。由于我们成功地使用独特的 FRT 序列操纵各种细菌基因组，而没有对基因组进行不需要的删除或重排，因此我们将扩展 Flp-FRT 的使用，以针对布鲁氏菌和伯克霍尔德氏菌物种开发具有耐汞 (MerR) 的新型遗传工具，例如i) 我们将开发和测试新的布鲁氏菌供体和克隆菌株以及穿梭载体（流产布鲁氏菌、羊种布鲁氏菌和布鲁氏菌）。 suis）和伯克霍尔德氏菌（鼻疽伯克霍尔德氏菌和假鼻疽伯克霍尔德氏菌）。将使用的两个非抗生素选择标记是 Asd（天冬氨酸-半醛脱氢酶）和 MerR。 ii) 我们将开发能够进行重复轮等位基因替换和转座子诱变的 FRT-MerR-FRT 盒。这些 MerR 盒的有用性将在流产伯氏杆菌和鼻疽伯克氏菌的等位基因替换实验以及伪鼻疽伯克氏菌的转座子诱变实验中得到证明。