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

Ohio State University, Columbus OH

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Abstract

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.