POWRE: Strategies for the Construction of Knock out Mutants in Hyperthermophilic archaea

该项目的长期目标是确定允许超嗜热菌在接近水沸点的温度下生存的分子机制。这些研究的基本假设是，在超过 90oC 的温度下生长需要存在共同调节的 DNA 修复基因。在之前的研究中，序列分析、生理学和生化研究已被用来研究极端嗜热菌的 DNA 修复过程和基因组完整性的维持。尽管这种方法非常成功，但越来越明显的是，必须开发新技术来了解嗜热菌如何防止其 DNA 热降解。这项工作的目标是建立破坏超级嗜热菌基因的遗传方法。这些方法广泛用于确定细菌和真核生物的基因功能，但尚未应用于超嗜热古细菌。为了实现这一目标，首席研究员将访问 Forterre 博士位于巴黎的实验室，并接受古细菌载体操作和嗜热菌转化方案的培训。首席研究员将利用从比较基因组分析中获得的信息来设计穿梭载体的选择性标记。将使用两种策略来完成基因破坏、体外转座子诱变和敲除已知基因的突变体构建。通过第一个策略，新的 DNA 修复突变体将被识别和表征。第二种策略将产生确定先前鉴定的修复基因的体内功能所需的信息。将研究突变表型对 DNA 损伤剂的敏感性。将确定被破坏基因的位置和身份，并分析其在接受诱变处理的细胞中表达的调节。该 POWRE 奖项将使 DiRuggiero 博士能够通过学习新技能和方法来开发独立的研究路线。

The long-term goals of the project are to determine the molecular mechanisms that allow hyperthermophiles to survive at temperatures near the boiling point of water. The underlying hypothesis of these studies is that growth at temperature in excess of 90oC requires the presence of co-regulated DNA repair genes. In previous studies, sequence analysis, physiological, and biochemical studies have been used to investigate DNA repair processes and maintenance of genome integrity in hyperthermophiles. Although this approach has been very successful, it is increasingly clear that new techniques must be developed to understand how hyperthermophiles prevent thermodegradation of their DNA. The goal of this work is to establish genetic methods for gene disruption in hyperthermophiles. These methods are widely used to determine gene function in Bacteria and Eukarya, but have yet to be applied in hyperthermophilic Archaea. To achieve this goal, the Principal Investigator will visit Dr. Forterre's laboratory in Paris and receive training in manipulation of archaeal vectors and transformation protocols for hyperthermophiles. The Principal Investigator will use the information obtained from comparative genome analysis to design selectable markers for shuttle vectors. Two strategies will be used to accomplish gene disruption, in vitro transposon mutagenesis and knock out mutant constructions of known genes. With the first strategy, new DNA repair mutants will be identified and characterized. The second strategy will yield information necessary to determine the functions in vivo of repair genes previously identified. Mutant phenotypes will be investigated for their sensitivity to DNA damaging agents. The location and identity of the disrupted genes will be determined, and regulation of their expression in cells exposed to mutagenizing treatments will be analyzed. This POWRE award will allow Dr. DiRuggiero to develop an independent line of research by learning new skills and methodologies.