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-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 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.

Our Supporters

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.