DNA-based System

Future Work

Obviously it is not trivial to go from an idea to a fully-functioning product over the course of one summer. Therefore we have envisioned the next series of experiments that would be performed to develop our project into something that was suitable for clinical trials, incorporating the ideas of constant feedback from modelling to wet lab and vice versa to ensure an optimal system. We propose five design-build-test cycles.

Design-Build-Test 1 - Full experimental proof-of-concept

We add IPTG to induce the production of TetR, which binds to the tet operator. We would test this by looking at the level of induction needed from our construct to reduce the fluorescence. This would allow a more accurate figure for our modelling of the amount of TetR needed and could be put into our model to see if this changes any parameters going forward.

Once we have established the amount of TetR needed, we would create cells as a double transformation with a TEV plasmid, and induce expression of TEV with arabinose. TEV can then cleave the TetR.

If TetR is cleaved then YFP will start to be produced.

We can relatively quantify the amount of YFP produced, comparing different levels of induction of TEV.

Design-Build-Test 2 - Proof-of-concept system in cell lysate

Instead of inducing TetR production, we add a calculated amount of purified TetR to the system and allow it to bind. This will check that our purification doesn’t affect the efficacy of TetR.

We induce the TEV protease by the addition of arabinose, as before.

TEV protease can then cleave TetR, as before.

We can quantify the fluorescence and compare all of the data to the system in the cells, and use this to feed into our DNA system modelling. We can also optimise our lysate.

Design-Build-Test 3 - Test with cruzipain and production of TEV protease

We would then construct a plasmid with TEV protease under the tet operator and promoter, and add this to a cell lysate with an appropriate amount of TetR, as determined in the above experiment cycles.

We would then add cruzipain to our system, to check that the TetR can be cleaved by cruzipain in the same way as it was cleaved by TEV protease.

The output would be the TEV-mediated cleavage of something like a quencher-fluorophore system, such as BBa_K2450501.

If cleavage occurs, then all the above steps were successful, and we will prove that the system works for cruzipain as an input.

Further DBT cycles

We would then repeat again, starting with a freeze-dried kit. This would check that it will be able to travel in this form, and that the addition of blood will be sufficient to rehydrate the kit. At this point we can also experimentally test to see the level of cruzipain which we can detect, and see if it matches our model.