In vivo study of gene regulation and gene function in Sulfolobus ssp.: application and development of transformation vectors

Sulfolobus是考古界研究得最好的高温古菌之一，已经发展成为一种重要的模式生物，特别是在了解古生菌的信息处理(即复制、转录、翻译)方面。然而，对于Sulfolobus和其他嗜热者来说，还没有有效的遗传系统可用。我们的实验室最近所做的努力，包括对Sulfolobus质粒的鉴定和开发稳定的、可选择的载体系统，为研究体内的基因调控和蛋白质/蛋白质相互作用以及改进和开发遗传工具开辟了新的途径。我们将使用我们的新型载体系统在体内表征受调控的启动子，并用于标签蛋白的表达。此外，我们将改进转化系统，使其能够在Sulfolobus中进行高效克隆，并希望开发用于突变和/或基因沉默的新工具，从而能够直接研究高温考古体中的基因功能。建议的方法包括利用脂质体开发新的标记和DNA摄取技术、辅助噬菌体系统、转座子突变和通过同源重组或RNA干扰进行基因沉默。

Sulfolobus represents one of the best studied hyperthermophilic archaea of the kingdom crenarchaeota and has developed into an important model organism in particular for the understanding of information processing (i.e. replication, transcription, translation) in archaea. However, no efficient genetic system is yet available for Sulfolobus and for other hyperthermophiles. Recent efforts in our laboratories including the characterization of a Sulfolobus plasmid and the development of a stable and selectable vector system have opened up new avenues to study gene regulation and protein/protein interaction in vivo and to refine and to develop genetic tools. We will use our novel vector system for the characterization of regulated promoters in vivo, and for the expression of tagged proteins. Furthermore, we will refine the transformation system such that it will allow high efficient cloning in Sulfolobus and we want to develop novel tools for mutagenesis and/or gene silencing that will allow the straight forward study of gene functions in a hyperthermophilic archaeon. The proposed approaches include the development of novel markers and DNA uptake techniques with liposomes, a helper phage system, transposon mutagenesis and gene silencing by homologous recombination or RNA interference.