A Model Archaeal System: Genetic Tools and Resources for T. kodakarensis

模型古菌系统：T. kodakarensis 的遗传工具和资源

• 批准号: 8535171
• 负责人: JOHN NEWTON REEVE
• 金额: $ 28.59万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8535171
• 关键词: Affinity; Affinity Chromatography; Amino Acids; Animal Model; Archaea; Biological; Biological Models; Biology; Biomedical Research; Cell Density; Cell division; Cells; Communities; Complex; Cytokinesis; DNA Repair; DNA biosynthesis; Development; Disease; Epitopes; Essential Genes; Eukaryota; Eukaryotic Cell; Experimental Models; Gene Expression; Gene Library; Gene Targeting; Genes; Genetic; Genetic Information Processing Pathway; Genetic Techniques; Genetic Transcription; Genome; Genomics; Health; Health Services Research; Hemagglutinin; Histidine; Homologous Gene; Human; Human Biology; Internet; Investigation; Laboratories; Libraries; Liquid substance; Mass Spectrum Analysis; Modeling; Molecular; Molecular Biology; Molecular and Cellular Biology; One-Step dentin bonding system; Open Reading Frames; Palpable; Plasmids; Procedures; Process; Prokaryotic Cells; Proteins; Recombinants; Regulation; Relative (related person); Research; Research Personnel; Resources; System; Thermococcus; Transcript; Translation Initiation; Work; genetic resource; genome sequencing; in vivo; knockout gene; programs; protein expression; research study; systems research; tool; vector

DESCRIPTION (provided by applicant): Research on bacterial nnodel systems has established much of our understanding of basic molecular biology. Extrapolations from bacterial systems to eukaryotic molecular biology and human health issues are however inherently limited by the lack of bacterial homologues of many components conserved in eukaryotic cells. Fortunately, Archaea do have homologues of many of these "eukaryotic" components and research with such simpler archaeal systems can therefore be legitimately extrapolated to eukaryotic/human cellular and molecular biology. The archaeal machineries, for example, that catalyze DNA replication and repair, transcription, transcript and protein processing, translation initiation, cytokinesis and cell division are all far simpler than their eukaryotic counterparts, but most of their components are proteins that have well-conserved structural and likely functional eukaryotic homologues. For experimental research, the attraction of these much simpler but eukaryote-homologous prokaryotic systems is obvious and palpable, but with very limited genetics and no archaeal model system, access of the research community to the advantages and opportunities afforded by the Archaea has been effectively blocked. We have recently established the genetic techniques needed for Thermococcus kodakarensis (T.k.) and, as solicited by the PA-07-457 request "...to develop and distribute genetic and genomic resources for emerging non-mammalian model organisms" the project proposed will generate the genomic resources needed to fully establish T.k. as a facile archaeal model system. Specifically, every protein-encoding gene will be Individually cloned and also modified to add a hemagglutinin (HA) epitope for Identification and a Hls6-afflnlty-tag for purification In sequence-verified plasmid libraries. T.k. strain libraries will also be constructed with every non-essential open reading frame (ORF) Individually deleted, and every genomic ORF HA-His6 extended. With the T.k. genetic techniques established and these genomic resources constructed, the first archaeal model system will be available. This will facilitate, expedite and encourage Investigations ofthe many archaeal cellular and molecular biology svstems that are homologues of eukarvotic/human systems and so valid models for healthcare research.

描述（由申请人提供）：对细菌节点系统的研究已经建立了我们对基础分子生物学的大部分理解。然而，从细菌系统到真核分子生物学和人类健康问题的推断本质上由于缺乏真核细胞中保守的许多成分的细菌同源物而受到限制。幸运的是，古细菌确实具有许多这些“真核”成分的同源物，因此使用这种更简单的古细菌系统进行的研究可以合理地推断到真核/人类细胞和分子生物学。例如，催化 DNA 复制和修复、转录、转录和蛋白质加工、翻译起始、胞质分裂和细胞分裂的古细菌机器都比真核生物简单得多，但它们的大部分成分都是具有保守结构和可能功能的真核同源物的蛋白质。对于实验研究来说，这些简单得多但与真核生物同源的原核系统的吸引力是显而易见的，但由于遗传学非常有限且没有古菌模型系统，研究界获得古菌所提供的优势和机会的机会已被有效阻止。我们最近建立了 Thermococcus kodakarensis (T.k.) 所需的遗传技术，并且根据 PA-07-457 请求“...为新兴非哺乳动物模式生物开发和分配遗传和基因组资源”的要求，该项目将产生完全建立 T.k. 所需的基因组资源。作为一个简单的古菌模型系统。具体来说，每个蛋白质编码基因将被单独克隆并进行修饰，以在序列验证的质粒文库中添加用于鉴定的血凝素 (HA) 表位和用于纯化的 Hls6-afflnlty-标签。 T.k.构建菌株文库时，每个非必需的开放阅读框（ORF）将被单独删除，并且每个基因组 ORF HA-His6 都会被扩展。与 T.k.遗传技术的建立和这些基因组资源的构建，第一个古菌模型系统将可用。这将促进、加速和鼓励对许多古细菌细胞和分子生物学系统的研究，这些系统是真核/人类系统的同源物，也是医疗保健研究的有效模型。