Friday, 16 May 2014

Project Tenebrio: larval proteome emerging from theY10s

This is just an update on our Tenebrio molitor meal-worm project. The last two sessions have focused on developing a robust extraction method for proteins and the application of ion exchange for identifying the most abundant classes of protein. We have decided that the traditional mortar and pestle is inadequate to the task! It proved difficult to obtain reproducibly high concentration extracts across the group, using freeze-dried larvae. We did however make significant progress using the slightly more technologically advanced glass homogeniser (kindly donated by Mel in the Department of Molecular Biology and Biotechnology at the University of Sheffield)! Using this method, and only two larvae (and importantly keeping everything cold on ice) we have been able to obtain extracts in about 30 minutes (following centrifugation) that are suitable for downstream protein (and nucleic acid) purification.

We first asked if the tissue extracts could be resolved cleanly on SDS PAGE and then collected no salt, medium (500mM) salt and high salt (1M) fractions: then Michael ran a range of class samples on a 10% SDS gel. As a result we clearly identified two major species at high molecular weight one eluting at low salt and the other binding tightly at pH7.4 to the Q sepharose (kindly provided by our friends at Eden Biodesign). Dr. Mark Dickman in the Department of Chemical and Biological Engineering at Sheffield has agreed to submit his samples for Mass Spec identification. However I am really keen to identify the low molecular weight protein that eluted a high salt (see below left). It could be one of Tebrio's antifreeze proteins, which are of some considerable biotechnological interest! I will let you all know as soon as we get the results back.

The protein extraction and analysis is running hand in hand with our development of a data base for T. molitor. There are around 6 000 transcripts documented at NCBI and each of the Y10s has been allocated 100 each, giving us complete coverage so far. We are working on a student friendly data set, and we are looking for keen Studio School collaborators to help us develop a user friendly interface or App over the coming weeks. This week we shall concentrate on improving the separation with larger columns and analysis of the nucleic acids. Keep up the nice work and we should soon be able to put together a manuscript for publication from the Y10 Class and Greenland Biodesign. I shall give you an update before half term and the data will be available to students on Edmodo.

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