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

该项目的长期目标是确定让嗜热微生物在接近水沸点的温度下存活的分子机制。这些研究的基本假设是，在超过90摄氏度的温度下生长需要存在共同调节的DNA修复基因。在以前的研究中，序列分析、生理和生化研究已经被用来研究DNA修复过程和维持高温菌基因组的完整性。尽管这种方法非常成功，但越来越明显的是，必须开发新的技术来了解超嗜热菌如何防止其DNA的热降解。这项工作的目标是建立基因破坏的基因方法在高温嗜热菌。这些方法被广泛用于确定细菌和真核生物的基因功能，但尚未应用于高温古生菌。为了实现这一目标，首席调查员将访问Forterre博士在巴黎的实验室，并接受有关操作古生菌载体和高温细菌转化方案的培训。首席调查员将利用从比较基因组分析中获得的信息，为穿梭载体设计可选择的标记。将使用两种策略来完成基因破坏、体外转座子突变和敲除已知基因的突变构建。在第一个策略中，新的DNA修复突变体将被识别和鉴定。第二种策略将产生确定先前确定的修复基因在体内的功能所需的信息。将研究突变表型对DNA损伤剂的敏感性。将确定被破坏的基因的位置和身份，并分析它们在暴露于诱变处理的细胞中的表达调节。这一POWRE奖项将允许迪鲁杰罗博士通过学习新的技能和方法来开发一条独立的研究路线。

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.