Outlook

Nanobodies

The nanobodies have not yet been introduced to our construct. The amber codon is in our plasmids, however, we had problems during our molecular cloning of our helper plasmid. Only after accomplishing that we can start expressing the non-canonical amino acid incorporating ferritin constructs.

Then, we can start the purification and proceed doing experiments for click-chemistry and characterizing the transFERRITIN system.

Here's what we have planned in the lab:

Here's what we have accomplished in the lab:

The apparently successful GGA product needs to be re-transformed into bacteria culture. Afterwards, we can try the double digestion again to double proof the correct insertion of our fragments. If this is successful, we can continue with the co-trafo of both of our plasmids and try if our nanobodies can bind to our ncAA on the Ferritin L-loop. If they do so, all of our parts are complete and we can undergo research on their selective target penetration and cargo release.

Once the proof of concept with GFP works, the next plan would be to work with a nanobody that would target a surface molecule on a bacterium.

Alternatives

If the nanobodies prove to be unsuitable we have a few other targeting molecules that we can take into consideration:

1. Pattern recognition receptors (PRR) proteins
2. Complement receptors

To lock drugs within the ferritin, modification is necessary to adjust the specificity for drugs within the ferritin.

Quercetin, Rutin, Flavone, PHB ester

We conducted an agar diffusion assay on E. coli to test the herbal components supplied by SjF Hanse Scientific GmbH. Despite our best efforts to optimize the test, we were unable to achieve a consistent, inhibitory effect on E. coli.

We reached out to Hanse Scientific to discuss possible solutions, and they suggested that we also test the components on a Gram-positive bacterium as they had shown a stronger effect on such bacteria. Additionally, they advised us to use a liquid medium instead of agar diffusion for the test, as it was more compatible with the components.

Therefore, after the iGEM competition, we plan to conduct further testing of the components in liquid medium and measure the inhibitory effect on bacterial growth through photometric measurement.

Alternatives

If these substances prove to be unsuitable, we have a few other cargo options that can be taken into consideration:

1. mRNA
2. Antibiotics
3. Recent research suggests that synthetic compounds can inhibit antibiotic resistance. Encapsulating these compounds with specific antibiotics can reactivate their effectiveness
4. Overload of iron to cause a toxic effect in the bacterial cell
5. Iron ligand drugs

Penetration

Our penetration assays have not been satisfactory. Either we will need to troubleshoot and optimize or find different CPPs with higher penetration efficiency.

Cytotoxicity

We need to test if the CPPs trigger cytotoxicity in human cells or trigger an immune response.

Modulation

For a higher modulation, the conjugation method of CPP and ferritin can be adjusted. Various Pathogens could be susceptible to different CPPs. In an emergency, a swift change of bound CPP could save lives.

Possible methods might include chemical inducible dimerization or the tag/catcher technology if the catcher can be reduced in size.

Alternatives

If the CPPs prove to be unsuitable, we considered the use of defensine as an alternative.

Dis- & Reassembly

The pH-dependent Dis- and Reassembly of ferritin has been documented thoroughly, yet we have not tested it ourselves.

With transmission electron microscopy (TEM) we can obtain high-resolution images of ferritin. By changing the pH to extreme values we want to document its disassembly followed by a reassembly by returning the pH to neutral conditions.

This step has to be done with all of our ferritin constructs to ensure we can control the encapsulation of substances in vitro regardless which cell-penetrating peptide we fuse to the subunits of ferritin.

Encapsulation

Followed by the tested assembly mechanisms we will conduct our own encapsulation experiments.

This step has to be done with all of our ferritin constructs.

Purification

Our purification has proven to be challenging, while we have successfully purified our WT-Ferritin, we have not yet managed to obtain purified CPP-Ferritin constructs. Additionally, our WT-Ferritin yield has been low.

This step requires further troubleshooting for optimal results.

Alternatively, we have considered using His-Tag for purification, which we then cleave by TEV protease.

Further modifications

During the coming course of our further experiments, we might face new challenges or seek further optimization. A few possible steps that can be taken for optimization are listed here:

1. It would be conceivable to achieve the inclusion of the components by other, directed methods. One possibility here would be the cysteine-maleimide coupling, in which the ferritin is provided with cysteine residues on the inside, via which coupling of the active substances to the reactive thiol groups can take place.
2. We could optimize the encapsulation of components by changing the surface charge inside ferritin. We can use Rosetta for protein redesign. Prof. Dr. Tobias Beck has successfully changed the surface charge of ferritin while maintaining its folding and function.

Proof of Concept

Our developed construct has to be tested on pathogens. Our complete, loaded, construct would have to be tested and measured in its efficiency, selectivity, kinetics, etc.

After the construct is prepared and the penetration is successful, we will perform tests on the components both with and without transFERRITIN to compare their efficiency. If our hypothesis is confirmed and there is a noticeable increase in the effectiveness of the transport, we will proceed to the next steps: Among others, we will follow the advice of Jonas Ide and Dr. Thomas Grunwald to obtain investors for transFERRITIN and gain clinical approval for the product.

Disassembly

A question we have not yet answered is if our construct will be disassembled in our target cells.

Our hypothesis is that, as mentioned by experts such as Prof. Knobloch, the bacteria will willingly deconstruct ferritin, releasing its cargo. This hypothesis is based on the data that bacteria, when infected a person will seek out iron for its own development causing deficiencies in its host.

However, this will have to be tested.

In case this does not happen, we plan to include a protease binding region that will be degraded by a protease within the bacterium.

Prof. Tobias Beck reminded us that the choice of the disassembly strategy affects the inclusion method. Covalent binding would not be suitable for inclusion because the components would not be released during disassembly (Verlinkung Gespräch Beck).