Final year projects
Ongoing research in the Denecke team is in a constant flux due to the short term experimental strategies that give rise to new data, often requiring the formulation of new working models and further experiments to test these. Proposed projects are therefore subject to change, this is particularly relevant for the 40 credit laboratory-based projects. Applicants are strongly advised to speak to Prof. Denecke and his team members in person to find out more details.
A) Laboratory-based 40 credit projects
All projects are addressing important questions in plant cell biology that can be published in peer reviewed journals and the students will be fully integrated in the research team. This means they have to follow good laboratory practice, participate at the lab meetings and contribute to routine maintenance such as preparing media, racking tips and autoclaving tubes and washing up. You will gain a good perspective on real lab work and at the end of the project you will have a much better idea of what you might want to do in the future. The projects listed below are just a few examples and students are strongly advised to make an appointment to discuss options. Do not leave it to the last minute to choose your final year project, come and talk to us soon so that we find an interesting project for you.
1) Discover the secrets of endoplasmic reticulum import sites
Much is known about the vesicles that transport cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. COPI vesicles return selected cargo from the Golgi back to the ER, whilst COPII vesicles transport bulk flow and selective cargo to the Golgi apparatus. COPII vesicles bud at specific ER export sites (ERES) but it is much less established if similar specialised ER subdomains exist that receive retrograde COPI vesicles. In collaboration with the group of Prof D. Robinson (Heidelberg, Germany) we have found evidence suggesting that the ER exhibits specialised subdomains that may function as ER import sites (ERIS), and current ongoing research in our team offers the prospect of two final year projects.
Project 1a): Targeting signals of the syntaxin SYP81. Syntaxins are specifically involved in controlling the docking of transport vesicles and accumulate in the target membrane. But just like other proteins of the secretory pathway, they are synthesized on the ER surface and have to be transported to their final destination. In the case of SYP81, it does not have far to go, but it is still unclear which mechanisms specifically target this molecule to ER subdomains. The aim of this project is to establish if SYP81 contains specific retention or transport signals to accomplish this task. The work will involve recombinant DNA techniques with plasmids, working with E.coli and Agrobacterium tumefaciens as well as in vivo imaging of tobacco leaf epidermis cells via confocal laser scanning microscopy
Project 1b) Identification of SYP81-interacting proteins. Syntaxins work together with two other SNAREs at the target membrane to form a tetrameric SNARE complex with the arriving vesicle SNARE. In plants, this tetrameric complex is far from established and the aim of this project is to identify binding partners of SYP81 from a selection of candidate SNAREs. To carry out protein-protein interaction studies, we need to generate specific antibodies for immuno-precipitation reactions. The work will thus involve PCR cloning, recombinant DNA techniques to generate protein expression constructs and gene expression experiments in E.coli.
2) Sustainable biofuel production: self malting potatoes
Now that energy is becoming more and more expensive due to the slow depletion of fossil fuels, we have to start exploring renewable alternatives. Although potatoes represent a high yielding energy crop per hectar and per time of occupied land, they are currently not envisaged as an energy crop. This is mainly due to high production costs and the difficulty to convert starch to fermentable sugar in an economically feasible manner. This project aims at overcoming these limitations by establishing self-malting potatoes. The strategy is based on engineering endoplasmic reticulum stored hydrolase pools which do not make contact with starch until the rasping and mashing step. Our lab has been active in identifying efficient ER retention strategies to avoid saturation of the signal mediated retrieval pathway from the Golgi and identified several limiting factors, allowing again two possible final year projects. The work will involve site-directed mutagenesis, protoplasts transfection and transport assays to measure ER retention efficiency.
Project 2a) Improving true ER retention. COPII-mediated ER export involves selective and unselective transport. The latter mostly carries soluble cargo molecules that passively move as part of the fluid phase within the vesicle (=bulk flow). Since the starch hydrolases are soluble proteins secreted by the secretory pathway, addition of ER retention signals will lead to rapid saturation of the HDEL receptor. We have found that some sequences preceeding the HDEL signal improve ER retention. This project aims to establish if this is due to the presence of acidic residues which could form calcium bridges and thus limit diffusion and subsequent bulk flow. The work will involve site directed mutagenesis of green fluorescent protein (GFP) reporter fusions followed by fluorescence recovery after photobleaching (FRAP) experiments to measure mobility in the ER.
Project 2b) Improving true ER retention. Identical to 2a), but instead of using GFP-fusions, α-amylase fusions will be generated and quantitative protein transport assays as well as protein stability assays will be carried out.
B) 20 credit Literature projects
Literature projects are often underestimated by students who think that it merely involves writing a long essay. In reality, it is expected that literature project write-ups are at least as comprehensive as those of laboratory projects, and require broadly pitched introductions, moving towards the specific topic of interest followed by a very thorough review of all published peer reviewed research papers covering the biological question. Finally, the write-up ends with a project proposal suitable to take the field further. This requires thorough understanding of the revised literature, the methods behind the published experiments, and an analysis of conflicting reports in the literature, justifying further experiments. Students choosing this option are advised to arrange weekly meetings with Prof. Denecke to discuss progress as well as problems.
1) Are there really two vacuolar transport routes in plants?
The concept that plants may have two or more functionally distinct vacuoles within the same cell was introduced in 1996 and was extremely popular with plant cell biologists. Now some researchers feel that the model was built on weak ground and more and more published results seem to argue against such a scenario. This literature project is aimed at revising the literature regarding the subject of vacuolar or lysosomal sorting in plants, yeasts and mammals, and to critically evaluate all publications related to the two-vacuole hypothesis.
2) Where do the endocytic and biosynthetic routes of the secretory pathway converge?
The endoplasmic reticulum of eukaryotic cells is the first organelle of the secretory pathway and delivers proteins and lipids to the Golgi apparatus from where they proceed to their final location. The plasma membrane is the entry point for the endocytic route and leads to the so-called endosomes, a complex set of intermediate compartments that also plays a role in the biosynthetic Golgi to vacuole route. This literature projects aims at critically reviewing all published evidence describing endosomes in eukaryotic cells and the mechanisms by which soluble and membrane spanning proteins are sorted.
3) Can organelles of the secretory pathway form de novo?
What comes first, the chicken or the egg? This question is relevant for scientists interested in the biogenesis of organelles, such as the vacuoles. Is it possible to build an entire secretory pathway from just a cell with a genome and a nucleus, or is it necessary for any new cell to receive a full complement of secretory pathway organelles from the parental cell line? Unlike the first 2 titles, this is a difficult project that requires excellent understanding of cell biology and is only recommended for first class students.
4) Biofuel: can it be sustainable and where will it make a difference?
Global warming is in the news every day, but few articles or reports about carbon footprints and bio-fuel production strategies are scientifically sound and well founded on measurable facts. The task of this project is to critically evaluate the literature on this subject and discuss if we really have to worry about man-made global warming and if we really can produce bio-fuel from plants in a sustainable manner. Again, this is a highly challenging project that requires excellent skills in critical reading and data analysis, and is again only recommended for first class students.