Team:MADRID UCM/Improving

Improving – bueno – iGem Madrid

Brook Preloader
Wave Wave

IMPROVING

Our technology is rooted in the work of two former iGEM teams: iGEM INSA-Lyon 2016 and iGEM Madrid-OLM 2018

Technological progress traditionally follows a “sink or swim” logic that has brought us this far. And this does not seem likely to stop in the near future. We want this project to be our contribution to the progress stream, not by casting aside the old, but building upon previous research that has provided a solid basis for us to take the baton and keep going.

1 INSA-Lyon 2016

Lateral Flows & Membrane Adhesion

The iGEM team INSA-Lyon 2016 worked on the development of sensor technologies of lateral flow. Despite their great characterization of the holding and detection systems that were meant to be used, it was not possible to develop the prototype due to migration problems with the caption particles during the trials.

Our team has made use of the Lyon team’s protocols for the preparation of caption particles. We have taken this as a starting point and expanded on it with a protocol for aptamer conjugation to modified carboxyl surface nanoparticles, testing their liability to migrate through a membrane.

Likewise, we have standardized a trial system for the verification of the different systems that play a role in the lateral flow assays. The implementation of microfluidics in the membrane allows us to improve control of the flow in this kind of trial. Moreover, it brings along the possibility of designing multiple experiments that allow us to gather further data.

Aptamer folding

The INSA-Lyon team developed in 2016 their own aptamer-folding software, using the ViennaRNA and Rosetta tools. Their results regarding aptamer folding were highly satisfactory, and so we made this software the basis for our own modeling.

Regarding the aptamer folding, we have improved several features in the code. Specifically, we identified the weak spots of Lyon’s code (particularly the fact that it sometimes did not compile) and solved those errors in several ways, for example by erasing some columns that added default noise and messed up the scoring.

Furthermore, after working out the compilation-related issues, we added several optimization tools that reduced execution time and computational cost, such as Threads, by using several terminals or erasing certain features that had no real use for our goals. The result is a final code three times faster than its predecessor. Additionally, we managed to make our code compatible with both Linux and macOS.

To sum up, we have greatly improved the Lyon code for aptamer folding and implemented an AI to improve outputs. We managed to end up with an efficient code that creates new aptamers in only ten minutes. We have taken the Lyon code and re-designed it according to our needs in order to improve the results.

2 Madrid-OLM 2018

The Madrid-OLM 2018 team developed and created a functioning SELEX protocol for the first time in the history of the iGEM competition. A lack of time prevented the team from getting a fully developed aptamer by the end of the competition, yet they proved their method useful and able to successfully select related aptamers.
After digging into the documentation of the process and the obtained results, and following the feedback given by two former Madrid-OLM team members, we determined that the main point to improve was the lack of reproducibility.
The system of nitrocellulose columns that the Madrid-OLM team used, while cheap and fast, was closer to handcrafts than to science. It took a long time for the team to acquire the skill level necessary to succeed in building the membranes, and a single mistake could destroy months of lab work. It was definitely not a tool that could be used by other iGEM teams to develop their own aptamers.

We decided that our objective this year would be the improvement of this protocol, by standardizing it and making it reproducible.

How? By robotic automation
Hence, part of our project involved working on the automation of last year’s protocols by an OT-2 pipetting robot and a number of modules specially designed to carry out a robo-SELEX process.

Even though our final result is not a fully robotic SELEX (as this requires resources beyond the reach of an iGEM team), over the course of this year we have obtained positive results that indicate our success in the automation of the four critical steps of SELEX.

In this way, we can replace the human agent with an OT-2 machine, an affordable lab robot that is standard for the iGEM competition. By following our process of fabrication of the different modules, documented on our website, any iGEM team can run a SELEX round that will take only a few days.

We hope that we have succeeded in making the process of discovering aptamers simple and reproducible , releasing future iGEM teams from the arduous task of doing it manually and, therefore, allowing them to focus on the development of new applications for the aptamers that the OT-2 will discover for them.