Genome scale genetic tagging and protein localisation in Trypanosoma brucei . (360G-Wellcome-108445_Z_15_Z)

Our objectives are: 1) Acquire localisation data for the majority of the proteins expressed in Trypanosoma brucei 2) Curate the localisations using established keywords to construct a searchable database accessible through TriTrypDB 3) Assess the morphological phenotype of the cells expressing the tagged proteins 4) Create an interactive image viewer of the localisation database based on the Human Protein Atlas (http://www.proteinatlas.org/) 5) Enable the development of targeted, sophisticated smart assays to interrogate complex biological questions This project is made possible because of a new, tractable PCR-based genetic tagging technology. An amplicon encoding an eYFP tag and a resistance gene is targeted to the desired locus for homologous recombination by sequences incorporated into the PCR primers. The resulting cell lines that express an eYFP fusion protein are imaged and the localisation of the fusion protein subsequently categorised. This is most effect ive way of producing this genome-wide dataset. The localisation database will be an extensively used resource that will facilitate and stimulate research in the trypanosome field and the wider parasite scientific community. This application is focussed, timely and well supported by both the UK and the international research community.