Team:Oxford/future experiments

Future Experiments

Overview

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.

IL-10 system design-build-test cycles

• Part proof of concept (test each part and show correct activity)
• Whole system proof of concept (show everything works together and choose most effective hydrolase)
• Optimise promoter strength for functional RNA, pSoxS, length of sRNA and riboswitch affinity (find combinations which give highest sensitivity and largest response)
• Test system with a gut-on-a-chip
• Animal models

Tetracycline kill switch design-build-test cycles

• Maximise efficacy of the inducer molecule (high throughput screening to find a tightly binding analogue which is not known to be toxic)
• Optimisation of system sensitivity (maximise TetR binding affinity for inducer, optimise TetR expression and binding affinity to the promoter sequence)
• Verify the low antibiotic activity (show that gut microbiota are insignificantly impacted by the doses used)
• Show lack of toxicity to cultured gut cells
• Animal models

The first stage to improving the inducible kill switch would be the optimisation of the sensitivity of the device using high throughput screening.

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.

A key requirement for an effective kill switch for probiotics is high selectivity.