Outlook

In our project, we have seen many different applications and methods of characterizing our ELP hydrogel both inside as well as outside of cells. We can conclude that we have been successful at slowing cell division and that we can co-express other proteins together with our hydrogel. Furthermore, we have seen that the interactions are reversible at low concentrations from the UV-Vis experiment, but not at high concentrations when we form a hydrogel. This was unexpected, but could be due to impurities left in the mixture because of imperfect purification steps. We have also discovered more applications for our proteins than we had initially expected. The ELP hydrogel could for instance be used as an injectable hydrogel that slowly releases drugs over time.

However, there is still room for improvements in the future. First of all, the expression of IL-10 has not been very successful yet. This is one of the first things that we want to get working in the future, because then the therapeutic application of our cells can really start to be tested. It would also be interesting if we can implement a way where we can get more control over when both types of proteins are expressed. We want to do this by optimizing the use of different promoter sequences.

Other factors that are beneficial for our therapeutic application have come forward in our Human Practices conversations and we would like to develop these aspects further as well. We would like to optimize the freeze-drying protocol to get higher cell survival rates. When this is possible, cELPro can be made into a pill that can easily be stored. Furthermore, we would like to investigate other hosts such as yeasts or other bacterial strains to make cELPro even safer for the patient. More about this can be read in our optimization section on the Implementation Page. In some conversations with RIVM and VIG, it also came up that it is important to know the lifespan of the bacteria. This is something we want to clarify as soon as possible too. We intend to do this with a protocol for the CCK8 assay where we measure over the course of multiple days.

Even though our results look promising until now, they are not conclusive enough to really answer some of the questions we had. For instance, at the moment, we are still unsure if a fraction of the cells really stop dividing while the other fraction continues dividing, or that the whole population is still replicating but at a slower rate. Further experiments are needed to really conclude whether the cells containing a hydrogel stop dividing and if all bacteria in the culture contain enough of the hydrogel.

To find out more about the effects of the ELP hydrogel, in the future we would also like to test not only ELP triblocks, but also use other controls such as diblocks to see what kind of effect this would have on cell division. In this case, we would expect micelle formation instead of hydrogel formation, which will likely give a different effect. We would also like to test more conditions such as different pH levels since this will also help to find more applications for cELPro. We also want to test whether our bacteria would have a higher survival rate when exposed to oxidizing conditions, since IBD is known to cause oxidative stress in the gut^[9][10].

In conclusion, we were able to gather promising results during our project and there are still plenty of options to improve the project in the future. We are curious about what the future will bring for cELPro!