Attributions; what the Aberdeen iGEM team members did

Alpha Team – Ag43 Protein

Team Members: Ana Maria Cujba and Martyna Sroka carried out the following work;

Alpha team took an Ag43 part produced by the 2012 Hokkaido University iGEM team, the goal at first appeared simple – insert a FLAG-tag-multiple-cloning-site (FLAG+MCS) into Ag43. However, two further problems had to be overcome first: 1. The part had six additional Pst1 sites (non-compliant with RFC-10) which did not appear in the registry sequence. These sites had to be removed. 2. Ag43 has beta hairpins, these are somewhat like Velcro and stick the cells together so that can form biofilms, for our assay these would lead to false-positives and two of them had to be removed. Only after solving those two problems, alpha team proceeded to insert FLAG+MCS; they also experimentally characterized all Ag43-related parts.

Omega Team – INP

Team Members: James Long and Joseph MacKinnon carried out the following work;

Omega team took an INP part produced by the 2011 Edinburgh University iGEM team, (this part is simply INP with engineered yellow fluorescent protein (eYFP) attached to the C-terminus). A FLAG(and HexaHis "His")-MCS had to be inserted onto the C-terminus of eYFP, although this is simple in theory it still took longer than expected, not only due to PCRs misbehaving, but more importantly the antibody test was negative and it was suspected that the eYFP (having its C and N termini on the same side) was shielding the FLAG/His tag. Because of this, the eYFP had to be removed and the FLAG tag attached to the C-terminus of INP.

Modelling and GFP Detector

Team Member: Konstantin Gizdov carried out the following work;

Mathematical modelling proved to be extremely useful to the team, it flagged up that an ELISA assay would probably not work, and as such when this gave poor results it was immediately abandoned in favour of a magnetic pull-down assay. It was also able to predict what the optimum ratio of “sender” to “receiver” cultures would be to give the best signal-to-noise ratio. As the ultimate goal of our project was to deploy a HAT detection kit in Africa, a cheap GFP detector would be needed, the only ones on the market are highly precise and expensive. A working DIY detector was created from off-the-shelf parts that cost less than £100 total and could be powered by a cheap solar-cell.

Parts and Quorum Testing

Team Member: James McAvoy carried out the following work;

This task can be split into three components. 1. Testing the individual expression of FLAG by Ag43 and INP use of anti-FLAG antibodies and fluorescence microscopy to see whether the plasmids worked. 2. Producing the composite cells (with Ag43 or INP plasmid, sender or receiver plasmid, and RFP identification plasmid all in the same cell). 3. Quorum-testing was a data-intensive task, these two cell cultures were tested with anti-GFP and anti-FLAG antibodies. At first ELISA-type assays were used to achieve this but these produced very poor results and were upgraded to a magnetic bead pull-down assay which were much better and gave us a proof-of-concept.

All submitted BioBrick parts were designed and created by our team:
• Bba_K1352000 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K1352001 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K1352002 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K1352004 (Jamie Long, Joseph MacKinnon)
• Bba_K1352006 (Jamie Long, Joseph MacKinnon)

All parts characterisation experiments were performed by the members of our team:
• Bba_K759001 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K542009 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K346007 (Martyna Sroka, Ana-Maria Cujba)
• Bba_K1090000 (James McAvoy)
• Bba_T9002 (James McAvoy)
The GFP detector was designed and built by Konstantin Gizdov.

Special thanks to:

• Dr. Liesbeth Van Nieuwenhove, for putting us in touch with Professor Philippe Büscher
• Professor Philippe Büscher, Head of the Unit of Parasite Diagnostics at Institute of Tropical Medicine Antwerp, for providing scientific expertise as well as supplying monoclonal antibodies and variable antigen type specific antisera
• Dr. Kenneth Skeldon, Rhiannon Thompson and Heather Doran, for their help with preparation for our presentation at PechaKucha, Explorathon - European Researchers Night event
• AU magazine, for publishing a series of blogpost about iGEM, synthetic biology and our project
• Edinburgh University iGEM 2014 team, for organizing a meeting with Professor Muffy Calder, Chief Scientific Adviser for Scotland, at The Royal Society of Edinburgh
• SULSA, for organizing Scottish iGEM teams meet-up and giving us a chance to present our project at the Synthetic Biology conference
• Kim de Moura, for his advice and support during Scottish iGEM teams meet-up event
• Kevin MacKenzie, for fluorescence microscopy training
• University of Dundee, Sequencing Unit, for Sanger sequencing of our created BioBricks
• Kelly Reid, Alison Davidson and other members of lab support team, for their patience, optimism and willingness to help
• Dr Marco Thiel and the Institute for Complex Systems and Mathematical Biology, University of Aberdeen for proving us with specilised software support and initial building materials for the device (Arduino, RaspberryPi).
• Last but not least - our supervisors: Dr Susan Black, Dr Rey Carabeo, Dr Alessandro Moura, Dr Berndt Müller, Dr Stefania Spanò and Prof Ian Stansfield, for their patience, scientific expertise and input during brainstorming sessions

We would like to extend our thanks to the organizations and companies that supported us and enabled our team to participate in this year's iGEM competition.