To investigate the intracellular localisation of a newly identified protein is a central strategy in molecular cell biology. The cellular function of a protein depends on its localisation. The classical approach has been immunofluorescence, but this requires antibodies against the protein that are specific and in addition the cells have to be chemically fixed, which means that it is not possible to see dynamic changes of localisation (for instance in the course of cell division). The development of GFP-technology was therefore a great breakthrough because it became possible to follow the localisation of a novel Protein by life-cell imaging.
Your task will be to exercise this approach using a typical case study: in frame of a cooperation project with the company Phyton Biotech, funded by the Federal Ministry of Research and Education (BMBF), we investigate the possibility to use modular metabolic engineering to generate new compounds by plant cell fermentation. As a proof of principle, we investigate alkaloid biosynthesis in tobacco BY-2 cells. A key enzyme is the enzyme N-methylputrescine oxidase (MPO1, Genbank accession number AB289456). This enzyme was cloned and overexpressed as GFP fusion in tobacco BY-2. Your task is to write up a strategy, how to do this by GATEWAY cloning. This includes
- delivery of the coding sequence and the protein sequence
- design of GATEWAY primers
- provide the map, which destination vectors you will use for the localisation study and for the metabolic engineering
- estimation of the costs for the project (ignoring the normal molecular biology, just find out the costs of the kits you need)
- Here you find an introduction explaining the different steps and asking some important questions: unpublished material (protected by password). NOTE: the questions are meant to be discussed...
- Here you find an example for a similar study from our lab: Maisch et al. 2009
- Here you find the plant GATEWAY page of Ghent University - read the review by Karimi et al. 2002 (you need to log into the KIT net to get full access to the pdf). Here you also find the vector maps.
- Here you find the GATEWAY manual including information on the principle, the mechanisms, and the primers