Contribution

Here we have gathered all the parts of the project that we have contributed.

During our year in iGEM we have done and learned a lot, and we hope that all this work can be helpful and inspiring for future iGEM teams.
We have gathered all the parts of the project that we have contributed. We have done both modelling, and parts design as well as very extensive protocols for ribosome display that haven't been very easily available before. We have documented our design issues and done documentation of the GMO laws in Finland.

Parts

We have contributed a set of individual parts as well as composite parts. Our main contribution is the set of GFP DARPin parts, meant for both expressions and for ribosome display. Ribosome display is mainly used to test other molecule's affinity against a certain target. Here, the GFP binding DARPin can be used in ribosome display both as a control and as a “rehearsal” DARPin before actually starting the real experiments.

Part Design Troubleshooting

We have had very long struggles in the wetlab with a few fragments, and for future teams handling with the same parts might find these tips useful:

  1. BBa_K4159012: We had trouble getting good concentration from the PCR. After trying multiple different annealing temperatures, we would recommend doing the PCR twice. So using the PCR product from the first run as the template for the second. This helped us both save the DNA fragment that we had ordered from IDT, and gave us a very good concentration to continue with.
  2. BBa_K4159011: We had trouble with the cloning of this fragment into the target plasmid. Our first design of the fragment had little spacer between the promoters. So our first tip is to always have at least +20bp between the promoter or even more. Our other design of the fragment was overall the same as before but had then a 50bp spacer in between. This made the fragment overall a little bit longer, which can be helpful in cloning especially if you use a basic restriction site as the cloning method.
  3. BBa_K4159003: This spacer sequence had not been used in this combination of parts before, however, according to literature it should be long enough to be used in ribosome display. We recommend not to go shorter on the spacer for ribosome display, to ensure that the target amino acid chain comes far enough out from the ribosome. Some spacers are almost three times the length, but that might introduce complications with repeats Thus, it can be hard to get them ordered as ready-made fragments. We found this spacer sufficient and easy to order from IDT.

DARPin Design

Throughout our design, we came across several challenges. First, we were too ambitious and desired to test a massive DNA DARPin library (complexity around 1024) which turned out to not be viable due to the lack of financial support and time. Secondly, we now know how challenging it actually is just to hypothesise, design, and construct a couple of antibody molecules for validation experiments as well as optimising every single base pair or amino acid sequence.

3D structure of a DARPin molecule,5 pairs of helices that are connected
3D structure of a DARPin molecule of three internal repeats (IR) flanked by an N-Cap and C-Cap.

We would first predict the protein structure of our DARPins (see Fig.1) of interest to further explore their molecular characteristics and possible binding sites with the AlphaFold2 algorithm. Secondly, we predicted the protein-peptide docking in silico with the usage of several available software with the aim to investigate which selected and optimised DARPin sequences best bind to our peptide of interest. Finally, we aimed to validate experimentally our computational simulation results in the lab and a couple of ribosome display assays with NGS analysis to find DARPins with high affinity.

We did one run of the ribosome display and sent it to the University of Helsinki, Viikki, institute of biotechnology, for sequencing and then we could further analyse what type of sequence combination best worked and bound to our target peptide.

During our wet lab period, we also successfully expressed our “proof of concept” DARPin, the GFP binding DARPin, which we knew would bind to GFP. We also used the GFP binding DARPin as a control in our ribosome display.

To validate our DARPins in action, we planned to build a bioreporter that would emit a fluorescence signal whenour target peptide was present in the environment and binding to the cells. Thus, when the DARPin would be added it bind the signalling molecule and inhibits its binding to the cell. Hence, with no binding, there would not be a fluorescence signal measurable, or at least not a very strong one in comparison to the one without DARPins in the environment. The construct of the bioreporter was inspired heavily by last year's Team IISER, Kolkata, who did a similar bioreporter but for S. aureus. We were extremely impressed by their work and saw that their design worked well, so we relied on the knowledge they passed on from last year. Similarly, we hope that also our work this year can serve as inspiration or as a base for some projects in the next year's iGEM competition.

Conlcusion

Overall, we knew our project was very ambitious for the timeframe given, especially our wet lab schedule. We divided our project into three parts, Designing the library, performing ribosome display, and building the bioreporter. We are happy that we have succeeded to work on all parts, and that we will get results from the library design and the binding assay. We hope that we still have time to finish the control of getting a baseline signal from the bioreporter, to validate that it works. As this was a very small scaled library, we intended that this would be the proof of concept and the same idea could be done on a broader scale in the future with more resources. To read more about our final results please check out our results page !