Team:Oxford/future experiments

Full Width Pics - Start Bootstrap Template

Future Experiments


Soon into our lab work we became aware of how optimistic our project was. Although we have a great belief in our project, one summer is not enough time to complete all the experiments to fully characterise our device. Here we have listed our plans for future experiments that we would conduct if we had more time.

We have split our future experiments into the two key areas of our device: the IL-10 system and the kill switch.

IL-10 system design-build-test cycles

  • Part proof of concept - we will test each part and show correct activity. This will be

  • Whole system proof of concept - incorporation of all parts into a chassis to demonstrate that the whole system works together. This will allow us to determine the most effective hydrolase, allowing us to decide whether to use a periplasmic hydrolase or a membrane anchored hydrolase.

  • Optimisation of promoter strength for functional RNA, pSoxS, length of sRNA and riboswitch affinity. This data will allow us to find out which combination gives the highest sensitivity and largest response. The corresponding combination will be the most effective treatment and will be chosen to take to the next stage.

  • Test system with a gut-on-a-chip. A gut-on-a-chip is a tissue culture which simulates a functional organ. We will use this system to find out the effects of our probiotic on the intestinal tissue and to discover any indirect effects on the local immune system. We may modify our system in response to the results in order to maximise the specificity of our device.

  • Animal models - Andra Necula emphasised the necessity for animal models in future experiments in order to test the systemic effects of the device on the immune system. The use of human microflora associated (HMA) rodents is likely to be the next step and would form an essential component of pre-clinical data.

Tetracycline kill switch design-build-test cycles

  • Maximise efficacy of the inducer molecule. We will conduct a high throughput screening to find a tightly binding analogue which is not known to be toxic

  • Optimisation of system sensitivity. We will maximise TetR binding affinity for inducers, optimise TetR expression and binding affinity to the promoter sequence.

  • Verify the low antibiotic activity. This will show that gut microbiota are insignificantly impacted by the doses used allowing the treatment to be terminated with as little effect as possible on the rest of the microbiome.

  • Show lack of toxicity to cultured gut cells. It is an essential factor of our kill switch that our device has as little impact as possible on the rest of the microbiome, this needs to be fully characterised in order to progress to clinical trials and to ensure the patient is comfortable in using our product.

  • Animal models. Like the IL10 system we are aware of the necessity of the animal models but we have not had sufficient time to plan these.

Lactococcus lactis

Once shown that our E. coli chassis could be lysed in response to the selected tetracycline analogue as a proof of concept, we would transform the system into our desired probiotic strain of L. lactis and show that the cells remained sensitive to the inducer and the endolysin remained effective at breaking down the cell wall. Our whole device also needed to be tested in L.lactis, this is particularly important for the hydrolase component to find out whether this component is functional in bacteria that it hasn't been tested in. It is possible that there are other potential interactions in the different bacteria that we haven't accounted for or do not occur in the chassis we have been using in the lab.