Cycle 1 - Hydrogel

Learn

From the UV-vis measurements, we found that the transition temperatures of our ELP constructs were quite a bit lower than we had expected. Furthermore, we learned that the phase-separation behavior is reversible, therefore giving the ELPs Lower critical solvent temperature (LCST) behavior. The CCK8 experiments showed that bacteria which produced the proteins were more resistant to ampicillin and divided less. Lastly, the diffusion experiment indicated that the hydrogels are capable of slow release of small molecules, and potentially also for protein drugs. The rate of this release is dependent on the weight percentage of the gel and can therefore be finetuned.

Apart from this, we have learned a lot from this cycle. Both things that we would do differently next time are also things that we can take with us into the next phase of our project. First of all, it was a lot harder for us to clone the constructs with rapamycin binding domains. We are still not perfectly sure of the reason that this happened. Later, we also realized that these constructs might not be the best design for our application, since rapamycin is quite toxic if it is taken up by the human body. This is why we decided to only continue with the ELPs that had Leucine zippers at their end.

Next time we will make sure to troubleshoot differently when it comes to cloning and transformations. Also, we would include more control groups if possible. We would for example add groups where a non-reated type of protein is expressed to see the effects of protein expression, and we would include ELP diblocks that canot form hydrogels. We later realized that we could have added more and better of these controls than we have now.

Cycle 1 - Hydrogel

Test

We expressed all constructs in E.coli BL21(DE3). These were then purified with either organic solvent^[5] extraction, inverse transition cycling or both. The first real test was to see whether they could form a gel at different weight/volume %. This went very well and the results can be seen on our Results Page.

The other experiments we did to characterize the hydrogel both inside and outside of the cell were dye diffusion, CCK8, confocal microscopy, and UV-Vis spectroscopy. These experiments were all meant to further characterize our hydrogel and prove that the bacteria would stop dividing. We can conclude that there are significant differences between our bacteria and untreated bacteria. The results of these experiments can be found in more detail on our Results Page.

The results of our molecular dynamics simulation can be found on our Simulation Page.

Cycle 1 - Hydrogel

Build

After designing the proteins that we wanted to test, the next step was to come up with a way to express them. First, we needed to clone the correct DNA sequences in the desired plasmids in order to obtain our constructs. Luckily, our supervisors already had some ELP constructs in their inventory that we could use as our starting point. We used pET24(+) vectors containing either one of the following ELP constructs:

1. VPGIG_[60]-VPGAG_[3]VPGGG_[2][12] = I_[60]-A_[60]

2. (VPGAG_[3]VPGGG_[2])_[8]- VPGIG_[60] = A_[40]-I_[60]

3. (VPGAG_[3]VPGGG_[2])_[12]- VPGIG_[60] = A_[60]-I_[60]

After digestion of these ELPs, they were ligated to create I_[60]-A_[100]-I_[60] and I_[60]-A_[120]-I_[60], have been explained earlier as A_[100] and A_[120], respectively.

We continued digesting these plasmids along with the ones in which were located the gBlocks (leucine zippers and rapamycin binding domains) encoding the crosslinking regions. This resulted in a variety of constructs containing these domains on either side of the ELP. In the end, we were able to create all constructs, more about the lab results from this can be seen on our Results Page. We have added all of these constructs as parts in the parts registry as composite parts. Furthermore, all their building blocks can be found as basic parts in the registry. The full overview can be seen on our Parts Page. The table below includes all the composite parts that we made and used to test hydrogel formation. However, protein expression went considerably better with the A120 construct that had the Leucine zippers, and furthermore, we found in conversations with our supervisors that rapamycin could be quite toxic. This is why from this point on, we tested mainly the Leucine zippers.

Cycle 1 - Hydrogel

Design

Our first goal was to create an intracellular hydrogel. Furthermore, we discussed with our supervisors that it would be great to have a system where the interactions are reversible and easy to trigger. This way, there would be more control over the system and it will become easier to work with. In one of the groups at our university, researchers work with Elastin-Like Polypeptides (ELPs) that have thermo-responsive properties and an be used to form hydrogels. These types of proteins seemed pefect for our application. The next step was to figure out how to crosslink the system to make the interactions strong enough to form a stable hydrogel. We started evaluating different methods of crosslinking ELPs to form the hydrogel. Eventually after brainstorming with our supervisors, we came up with two methods that we wanted to try. We considered others as well, such as using a split NanoLuc system, but the expectation was that there would be too much background to measure a good signal in the end and that their affinities would be too low.

Both strategies that we wanted to try involved constructing a triblock ELP containing a (relatively) hydrophilic core sequence, and two hydrophobic outer sequences. These sequences can be seen below. Furthermore, it can be seen that they are abbreviated to A100 and A120. They are two sequences consisting of the same amino acids, but with different lengths of the hydrophilic middle part. To the end of each hydrophobic block, a crosslinking group is attached differing for both strategies. We wanted to include and test both methods to have a higher chance of succeeding.

A100:

A120:

Strategy 1: consists of ELP triblocks with complementary Leucine zippers at their ends. The specific Leucine zippers used have been tested by Gradišar et al.^[2] And furthermore we found a paper from Fernández‐Colino et al.^[3] where they made an ELP-based injectable hydrogel where also cross-linking Leucine zippers were used. Furthermore, the temperature responsive properties of ELPs and these Leucine zipper interactions also seemed appropriate for our application, since we wanted to first grow our bacteria and then only when the OD is high enough, trigger the hydrogel formation by increasing the temperature to above the transition temperature.

As a control, a linker containing only one of the two zipper domains on each end was also designed. The zippers of this control are not complementary, so they should add no extra affinity to the interaction of the proteins. Therefore, it is expected that this control will form a weaker hydrogel compared to the construct with complementary zippers.

Strategy 2: is made up of the same triblock ELP but contains different crosslinking domains. Namely, the rapamycin binding domains: FRB and FKBP12. We have tried to make all different combinations with the different domains on the zippers. Rapamyacin can be used as a molecular glue and stiches the two different binding domains together^[4], so we expected that upon adding rapamycin to the purified proteins, a hydrogel would form. Here we also added controls that had the same binding domains on each end, and in this case, it was expected that no hydrogel would form upon the addition of rapamycin.

The combination of these two strategies with the different lengths of the ELPs used, made it so that we ended up with 10 different constructs that we all wanted to try. Furthermore, you can see in the figure what the different constructs look like schematically.

Cycle 2 - Therapeutic

Learn

We are not sure if the expression of IL-10 was successful. This still needs further development in the future. We learned that it is not straightforward to express proteins with disulfide bonds in bacteria, let alone co-express them. However, we are happy that we were still able to do a co-expression with another protein to show that the concept works.

Cycle 2 - Therapeutic

Test

We purified our proteins with periplasmic extraction and ran this fraction on an SDS-PAGE gel. However, from this gel, we could not say for sure that IL-10 was present or at what concentration it was present. Therefore, we decided to perform an assay on the periplasmic fraction, namely the Lumit™ IL-10 (Human) Immunoassay. This is a fast and easy-to-use assay which is able to detect human IL-10 at concentrations as low as 4.9 pg/mL. However, the results of this assay were not clear and we are unsure if the expression of IL-10 was really successful. The results of everything that we tried regarding the therapeutic can be seen on this Results Page.

However, we did succeed at the co-expression of [A3G2]_[12] mNeonGreen. This shows that it is possible to co-express proteins in our cels with a hydrogel in them. We also used these cells to do FRAP measurements, more about this can be read on our Results Page.

Cycle 2 - Therapeutic

Build

The IL-10 gBlocks were restriction digested, as was the pBAD vector. After a ligation, the following constructs were created:

Cycle 2 - Therapeutic

Design

In order to treat IBD with our bacteria, we needed to incorporate DNA that could encode for a protein that reduces inflammation. From the conversations with stakeholders and by doing literature research, we found IL-10 to be a suited candidate to use as a therapeutic^[6,7]. We read this in multiple articles and IL-10 was also mentioned in multiple of our Human Practices interviews. We also looked at other options such as some small molecules, but from the Human Practices interviews, it became clear that IL-10 would be the best option. We found evidence that IL-10 could be expressed in E. coli BL21^[9,10]. In order to co-express this protein with our ELPs, we needed to use a vector that was orthogonal to the already used pET vector. To this end, we decided upon using a pBad plasmid, just as the one used for the expression of I_[60]-mNeonGreen. The latter protein could be used as a model protein since it would require a similar co-expression protocol as when we would want to co-express IL-10 and the ELPs. More about this can be read on our Results Page and more about the parts can be seen on our Parts Page.

IL-10 contains two disulfide bonds and those cannot form properly in the cytoplasm of E.coli. This is why it was important that the cytokine is located to the periplasm so that it can fold correctly. Therefore, an N-terminal peptide sequence was added. We used two different signal sequences that we had seen used in literature before, the Ompf sequence^[9] and the PelB sequence^[10].