Human Practices

At the very beginning, the question arose as to which antimicrobial agents we would encapsulate in the ferritin container. We are very grateful that in this matter we received the support of the company SjF Hanse Scientific GmbH (hereinafter referred to only as Hanse Scientific) under the leadership of Dr. Juri Smirnov. In addition to various laboratory services, the Hamburg-based start-up conducts research in particular on antimicrobial plant substances. Since 2020, Hanse Scientific holds a patent for an extraction process of the plant extract from the Japanese tree Sophora japonica . The company markets the purified and concentrated plant extract under the brand name Sofoxin which is an antimicrobial care product for animals and humans.

Since the team at Hanse Scientific is conducting research into optimization and the mechanism of action, they were also interested in our project and in whether the plant extract is more efficient when being transported inside the cell instead of being applied externally. Thus, we are pleased that Dr. Juri Smirnov and the team of Hanse Scientific have been available to us for advice throughout the entire project, have always supported us quickly and helpfully in emerging questions, and supported us with shaping our project goals. We would also like to thank Hanse Scientific for the donation of the extract compounds with which we were able to carry out our experiments.

Most important takeaways

Due to close cooperation and scientific exchange, we have been able to gain some insights into the research of the plant extract Sofoxin (more information can be read in the patent):

1. Antimicrobial effect

   The plant extract shows antimicrobial activity on various bacterial pathogens. Here, antibacterial efficacy tests were performed on ATCC* classified strains (Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus epidermidis, Escherichia coli ) and multidrug resistant strains (MRSA, VRE). Inhibition of bacterial growth (minimum inhibitory concentration) by the plant extract was demonstrated using the microdilution method and by changing the optical absorbance.
   *ATCC (American Type Culture Collection) is a nonprofit, global biological resource center and standards organization and the leading developer and supplier of authenticated cells lines and microorganisms.

2. The main components of the plant extract

   By NMR and MS, the following major components were detected in the extract, which are partly responsible for the antimicrobial activity:

   • para-hydroxybenzoic acid ester (PHB)
   • Rutin
   • Quercetin
   • Kaempferol
   • Flavone

3. Synergies

   In addition, synergies were found between the individual components. Several studies found that the components in combination showed a stronger effect than each component individually. The synergistic effect reduced the concentration required for inhibition. It is believed that the multi-component nature of the extract makes it more difficult for microorganisms to develop resistance to the extract. For this reason, we consider the use of the synergistically acting components for our approach with transFERRITIN to be useful as an alternative for antibiotics.

4. Stronger effect on Gram-positive bacteria such as MRSA

   Dr. Juri Smirnov told us that he and his team have found that the plant extract has a stronger effect on Gram-positive bacteria than on Gram-negative bacteria.

5. Reduction to selected components

   Even though the strongest growth inhibition can be achieved by the whole plant extract, Dr. Juri Smirnov recommended a reduced selection of components of the extract. The reason for this is that the composition of components in plant extracts can vary from batch to batch, and with the natural extract we would not have a standardized, controlled composition of components. Thus, we would not be able to make any predictions about the exact efficacy, which, however, must be guaranteed with regard to an application in humans. This statement also agrees with the recommendations of Prof. Dr. Wolfgang Streit and Dr. Thomas Grunwald.

   Based on the components with the strongest synergistic effect and with regard to the solubility of the components, Dr. Juri Smirnov and the team at Hanse Scientific recommended the following selection of components to us:

   • Flavone
   • PHB ester
   • Rutin
   • Quercetin

   Of these, flavone/rutin can be tested in a two-drug combination and flavone/rutin/quercetin in a three-drug combination due to strong synergistic effects.

6. Solubility of the components

   Another important issue was the solubility of the components. Both Dr. Juri Smirnov and Prof. Dr. Wolfgang reminded us that the components that are to be encapsulated must also be water-soluble in sufficient concentration for us to achieve an efficient effect.

   The plant extract and components have previously been dissolved in organic solvents. For our approach, the Hanse Scientific team conducted various tests on water solubility and investigated up to which concentration the respective components are soluble in water and whether or how the efficacy changes. The Hanse Scientific team shared the test results with us in a meeting and donated the individual highly concentrated components in ethanol, which we could further dilute with water for our purposes. In the event that the organic solvent was a hindrance in our encapsulation experiments, Dr. Juri Smirnov and the team at Hanse Scientific discussed with us other ways to improve solubility. Whether the drugs dissolved in ethanol can be encapsulated in a dilute water solution in our ferritin transporter remains to be tested. It is possible that modifications would still need to be made regarding the solubility.

To-do-list after the talk

The first thing we did when we received the compounds was to test whether we could reproduce the results in our laboratory. For this, we tested the inhibitory effect on bacterial growth using agar diffusion assay.

We tested all four components (rutin, quercetin, flavone, PHB ester) individually and in combination on E. coli. Unfortunately, we had problems reproducing the results with our protocol.

As a result, we communicated again with the Hanse Scientific team and received the following advice:

• On one hand, Dr. Juri Smirnov informed us that in their trials the components worked better on Gram-positive bacteria than on Gram-negative ones. So it could be that we would achieve better results if we used a Gram-positive germ instead of E. coli.
• Second, a member of the team helped us adapt our protocol and compared his protocols with ours.
• Lastly, Dr. Juri Smirnov noted that the agar diffusion assay may not be the best method to test the inhibition of bacterial growth. Since the components are not completely soluble in water, they would not be suitable for agar diffusion assays, which require water-soluble components. Instead, he suggested tests in liquid culture and checking the absorbance by photometric measurements.

Unfortunately, we no longer had the capacity to implement the advice before the Wiki Freeze. But we will implement it in the lab afterwards and integrate it into our project.

The inclusion of active ingredients in our drug-delivery system transFERRITIN is an essential part of our project. In order to test whether the components can be encapsulated in drug-delivery systems, we need the fully expressed and purified CPP-ferritin container. Unfortunately, we did not manage to encapsulate the components by the time of the wiki freeze, but we will continue to perform the experiments in the follow-up. This leaves the following tasks to be completed:

• Test the inclusion of the components in the CPP-ferritin container and check the inclusion efficiency as well as the composition of the components.
• Possibly further adjust the solubility of the components.
• If the components are successfully entrapped in the ferritin, we need to test whether the penetration of the pathogens works.
• If penetration is successful, we will test the antimicrobial effect of the components with and without transFERRITIN (we expect a stronger antimicrobial effect by transport into the bacterial cell than by external application).
• Comparative controls with antibiotics will be performed.
• To test whether the synergistic effects could possibly inhibit resistance mechanisms of resistant bacteria and reactivate ineffective antibiotics in their effect, we would perform a cross test on a resistant germ and administer both the antibiotic and transFERRITIN with the herbal synergistic components.

What we integrated into our project

Through Hanse Scientific and their research on the antimicrobial effect of herbal substances, we were inspired to use herbal components for our drug-delivery system and to find an alternative to classical antibiotic therapy.

• Due to the poor reproducibility of the effect of plant extracts as well as the lack of standardization regarding the composition of the individual components in the extract, we decided in consultation with Dr. Juri Smirnov to use a reduced selection of individual antimicrobial components instead of the entire plant extract.
• Based on the promising effect of the synergistically acting components, we aimed to encapsulate a combination of two to three components to achieve a more efficient antimicrobial effect.
• We will dilute the components dissolved in ethanol and water and test them for inclusion in ferritin. If there are complications, we will continue to be in close communication with Hanse Scientific to look for a solution.

We would like to thank Dr. Juri Smirnov and the team at Hanse Scientific for their constant and helpful support! They have made a significant contribution to the further development of our project and, in addition to theoretical input, have also supported us with material donations by providing us with the antimicrobial components. We greatly appreciate the interest in our project and the successful implementation, and thank them for their great cooperation!

Our drug delivery system, transFERRITIN, is designed to specifically penetrate bacterial pathogens and releasing plant antimicrobial agents within the bacterial cell to obtain an efficient antibacterial effect. So, already at the beginning of our project it was essential to deal with the question of how to ensure the uptake of our Ferritin container in our target organism and which bacteria are most likely to do this.

As the Head of the Department of Microbiology & Biotechnology at the Institute for Plant Sciences and Microbiology in Klein Flottbek from the University of Hamburg, Prof. Dr. Wolfgang Streit was an excellent contact to assess the potential challenges of our project idea. Prof. Streit is an experienced microbiologist, and he talked with us about resistant pathogens and made a selection of bacteria to focus on. He also gave us advice on how to proceed if we want to work with plant extracts.

Most important takeaways

After presenting our project to Prof. Streit, we discussed the following topics:

1. Bacterial resistances and uptake into bacteria

   Prof. Streit explained two aspects to us at the beginning:

   • There are some bacterial pathogens with strongly developed resistances such as Pseudomonas aeruginosa, Klebsiella pneumonia and Staphylococcus aureus.
   • Resistance develops over several years and becomes established. Some strains hardly can, or no longer be treated with antibiotics. The question arises whether the plant extract can circumvent the resistances and still show antibacterial effect.

   What we can use to our advantage would be the fact that bacteria that produce biofilms tend to compete for iron. Since ferritin stores and transports iron ions in normal metabolism, a ferritin loaded with antibacterial agents could be preferentially taken up by the bacteria in the biofilm to compensate for the iron deficiency. This could even support ferritin uptake for our purposes.

2. Antimicrobial plant extract

   Prof. Streit informed us that experience has shown that it is difficult to reproduce the effect of multifactorial plant extracts. The composition as well as the concentration of the individual components of the extract can vary from batch to batch. For a therapeutic approach it is necessary to always achieve the same effect with the same precise mixing ratio and dose.

   Since this variability (regarding the mixing ratio of the components and the concentration) and the reproducibility could be a problem for us, Prof. Streit recommended that we reduce the plant extract to a few components and use only the most active ones.

   We took Prof. Streit's advice seriously and then met again with the Hanse Scientific team to talk to them about a possible reduction of the whole plant extract to a few components. Finally, we found a selection of effective, synergistic components that we used for our approach.

   However, Prof. Streit gave us important advice at the end: Even if a reduction to a few components can be useful, Prof. Streit advised us to try our experiments also with the whole plant extract. Just because various problems with plant extracts have been encountered in other projects so far does not mean that we cannot try and possibly find a solution for our approach.

3. Solubility of the antimicrobial components

   Lastly, Mr. Streit pointed out the following thing to us: in order to encapsulate the antimicrobial components of the plant extract into ferritin, the components must be present in a water-soluble form. A good mixing ratio of the components is therefore essential.

   We also took these details with us into the next conversation with the team from Hanse Scientific GmbH.

To-do-list after the talk

1. Do research about the (specific) ferritin uptake of bacteria.
2. Talk to Hanse Scientific about a selection of components from the antimicrobial plant extract.
3. Make sure that the components are water-soluble and compatible to be encapsulated in ferritin.
4. Use antibiotics as a control to the plant components to compare the efficiency.

What we integrated into our project

• Since Pseudomonas aeruginosa is a biofilm-producing bacterium and has been classified by the WHO as one of the most critical resistant pathogens where urgent action is needed, we decided that we would focus on the P. aeruginosa pathogen after a proof-of-concept using Escherichia coli initially.
• Instead of using the whole antimicrobial plant extract, we talked to the team from Hanse Scientific GmbH and figured out, that a selection of four active ingredients would be suitable: quercetin, flavone, rutin and para-hydroxybenzoic acid showed synergistic effects and can be used in a water-soluble manner.

Finally, we talked to Prof. Streit about a possible cooperation with his research group. As helpful as he is, he offered us his further support and, if necessary, allowed us to carry out our tests on the pathogens in his laboratories. We would like to thank Prof. Streit for his helpfulness and support as well as his competent advice. He has thus laid an important foundation at the beginning of our project.

Our drug-delivery system relies on the use of the ferritin protein as a transport container. We aim to modify the surface of ferritin to allow for specific transport into bacterial cells, while also enabling the enclosure of various substances in its cavity. To ensure the success of our project, we consulted a ferritin expert at the start to gain valuable feedback.

We are grateful to Prof. Dr. Tobias Beck from the University of Hamburg for generously agreeing to be our discussion partner. He provided us with valuable insights from his research, alerted us to potential challenges in our project planning, and offered suggestions for potential solutions. We greatly appreciate his time and expertise.

Prof. Dr. Tobias Beck is currently conducting research on biohybrid materials that involve proteins and nanoparticles, as well as the production of nanoparticles using biotemplates. With a comprehensive understanding of structure elucidation and protein crystallography, Prof. Beck has also gained experience in computational protein redesign. He has informed us that he strives to combine the various areas of his scientific career and work in an interdisciplinary manner. One of Prof. Beck's research areas is focused on ferritin, which he has extensive experience with, including its use as a drug-delivery system. We have been fortunate enough to learn a great deal from his expertise and integrate it into our project.

Most important takeaways

1. Ferritin

   Ferritin is a protein that helps with iron metabolism by storing and transporting iron. This protein consists of 24 subunits that can assemble or disassemble depending on the pH value. Professor Beck mentioned another protein called encapsulin, which is also a container protein with a larger cavity but has a different way of including materials. If we need a larger volume inside the container, encapsulin could be an alternative. However, ferritin has its advantages too. It is biocompatible, can hold cargo, can assemble/disassemble in a pH-dependent manner, and has natural properties to enter cells like tumor cells via the transferrin receptor. But if we use ferritin as a drug delivery system, we need to carefully consider how to release the cargo at the target site and achieve disassembly in the bacterial cell. Professor Beck advised us to think about these issues from the beginning of the project to lay the groundwork.

2. Surface modification

   The protein ferritin can be modified through molecular biological techniques like cloning and mutagenesis. The transFERRITIN project aims to modify the surface of ferritin by attaching Cell Penetrating Peptides (CPP) to facilitate the transport in bacterial cells. Professor Beck, who has extensive experience in ferritin modification, shared valuable insights and advice from his research. Professor Beck and his team were able to modify the surface charge of ferritin without disrupting assembly or function by using Rosetta for protein redesign. Rosetta is a useful but laborious tool to redesign the protein.

   Surface charge is crucial in the uptake of ferritin into cells, as evidenced in a previously published paper by Prof. Beck on ferritin delivery and uptake in eukaryotic cells. However, since the project focuses on bacterial cells and CPPs, this information is kept in mind.

3. Assembling

   We had an important question regarding the assembly of ferritin subunits when fused with a CPP. We were uncertain if a single CPP per ferritin container would suffice or if we needed to clone multiple CPPs at each ferritin subunit. Our concern was whether the assembly of ferritin would continue to work efficiently with the addition of CPPs. We were advised by Prof. Beck that he knew of some studies where peptides were fused at the N-terminus of ferritin and the assembly continued to function. However, he recommended comparing and characterizing the assembly after each step using transmission electron microscopy (TEM).

   Prof. Beck also informed us that the ferritin subunits would assemble into the complex immediately after expression in the cell and would remain stable even at neutral pH during purification. An SEC analysis would show the peak at the 24mer.

4. Inclusion of components in ferritin

   An important aspect of our project was efficiently encapsulating our components in ferritin. Luckily, Prof. Beck, an experienced expert in this matter, provided us with the following points.

   • Whether the inclusion of a substance works depends initially on the size of the substance, since ferritin has only a limited cavity.
   • There are typically four methods of encapsulation.
     • Using reactive group of cysteines

       A strategy for introducing cysteines inside ferritin through point mutation has already been discussed in relevant literature. This creates a reactive group that can be modified for effective binding with the substance to be encapsulated. However, it is important to note that this would result in a covalent bond, which could pose challenges during the process of releasing the active substance as the bond would need to be broken.

     • Coupling of the drug via the reactive thiol group of the cysteine on the inside of the ferritin.

       To couple the drug to the ferritin, the cysteine-maleimide coupling was used here, in which the thiol group of the cysteine reacts with the maleimide to form a thioether. In this method, the ferritin must be modified so that the reactive thiol groups of the cysteine are located in the inner sphere of the assembled ferritin.

       The advantage of this method is that, assuming a successful reaction, the active substance has been taken up in the system in any case. In addition, other molecules, such as fluorophores, can also be introduced by this method. The disadvantage is that the target molecule must first be equipped with the maleimide residue. In order to release the drug, a mechanism for cleavage must be present. In the AG-Beck, an acid-cleavable hydrazone bond was used here.

     • Diffusion via existing channels in the ferritin

       Ferritin consists of 24 subunits, channels with a size of 3-4 Å can form within the intersections. Iron ions can diffuse through these channels, but also guest molecules. By lowering the pH value and slightly heating the solution, these channels can be easily enlarged. The disadvantage of the method is that the channel size also restricts the target molecule.

     • Encapsulation of the drug via pH-controlled disassembly and assembly

       Ferritin breaks down into subunits at both low and high pH values. The drug can be encapsulated by adding it during the pH change and returning to pH 7.

       According to the concept of statistical encapsulation, the substance is expected to encapsulate in ferritin in the same proportion as it exists in the surrounding solution. For this reason, it is important that the substances are soluble in water to allow for high concentration in the solution and subsequent encapsulation. To ensure success, it is crucial that the plant components used are water-soluble, as discussed in the Human Practices interview with Dr. Juri Smirnov and SjF Hanse Scientific GmbH . While statistical encapsulation may be an option, it may not provide the same level of efficiency as inclusion based on interactions. Prof. Beck advised us to consider the possibility of an interaction between the components and the interior of the ferritin, which could aid in confinement and improve efficiency.

   • If you want to modify or mutate the inside of ferritin, you can use the protein structure from PDB to identify the relevant and conserved amino acids.
   • Checking the quality and quantity of encapsulation is crucial to ensure that the components have been properly encapsulated. When using statistical encapsulation, it's possible for substances to bind non-specifically to the outer surface, leading to inaccurate absorption results. Therefore, it's important to verify encapsulation thoroughly.
   • Working with multiple components and substances instead of a single component requires precise controls and verification methods.
   • When considering encapsulation, it's important to think about how the cargo will be released at the destination. A covalent bond won't work because the components can't be released. Therefore, it's crucial to also consider how the container will be broken down in the bacterial cell, such as proteases that can degrade the ferritin container.
   • According to Prof. Beck, some substances can diffuse in and out at high temperatures because the pores widen enough to allow even large substances to pass through. By using Rosetta to redesign proteins and expand the pores, we may be able to bypass the need for the disassembly and release of components. Additionally, protein redesign could improve the interaction of components by altering the charge on the inside of the ferritin.
5. Methods and controls

   As previously discussed, it is crucial to establish procedures for regulating each stage of the process. Additionally, Professor Beck suggested considering analytics from the outset and implementing controls at every stage. It is beneficial to deconstruct our intricate system into specific components that we can evaluate. For instance, incorporating a labeling system can help us verify the number of encapsulated components and observe the successful infiltration of the CPP-ferritin container into the bacterial cell.

   It is unlikely that we can track the transport of the CPP-ferritin container using only our components. We expect to observe an effect once the components are released, but we need to validate the transport into the cell based on the observed effect. Prof. Beck suggested establishing the CPP-ferritin system by encapsulating nanoparticles and tracking the transport using negative staining with TEM. Pure ferritin has low contrast, so nanoparticles must be encapsulated. This method will allow us to check the localization in the cell and visualize whether the CPPs are functioning on the surface.

   An alternative method would be to use fluorophores (as described in the literature) that can be encapsulated and tracked using FACS. This would allow us to confirm the efficacy of the transport and the influence of CPPs on ferritin.

   Once we have established transportation, we can include our components instead of nanoparticles under the same experimental conditions. We would then expect to see the effect of bacterial growth inhibition.

6. Purification

   During the discussion, Prof. Beck emphasized the importance of having highly purified ferritin molecules without any tags like HisTag for his research. He explained that although affinity chromatography using HisTag is a common purification method, they opted for heat precipitation, ion exchange chromatography, and size exclusion chromatography to achieve the desired level of purity.

   We needed to confirm if high purity ferritin was required for effective assembly. Professor Beck provided us with reassurance by stating that ferritin begins to assemble right after expression in the cell and remains highly stable during the purification process. Therefore, we can expect the peak in SEC to be at the molecular weight of the fully assembled ferritin.

   Ferritin possesses a unique trait of being thermostable at temperatures up to 95 °C. This characteristic enables the purification process to be expedited through heat precipitation at the outset, which denatures the majority of proteins. Subsequently, ammonium sulfate precipitation causes the denatured proteins to precipitate, leaving ferritin to be further purified in the supernatant. It is plausible that SEC might suffice for our intended purpose to obtain a sufficiently pure ferritin.

To-do-list after the talk

After our helpful discussion with Prof. Beck, we created a comprehensive to-do list:

• After determining which plant components to encapsulate, the next step is to consider the best method for encapsulation. If the components are sufficiently water-soluble to achieve a high concentration in the solution, statistical encapsulation is a viable option. However, if the water solubility is insufficient, we can explore interactions between the ferritin interior and the components being used.
• In the same course, we must research the proper release of our cargo into bacterial cells, which affects our decision on encapsulation.
• After determining the encapsulation strategy, we must analyze the components to determine their level of encapsulation.
• To examine bacterial cell transport, we can use TEM negative staining with encapsulated nanoparticles.
• To purify ferritin, utilize heat precipitation as well as SEC and IEC techniques.

What we integrated into our project

During our conversation with Prof. Dr. Tobias Beck, we realized the need for further research and more detailed project planning before starting our laboratory work.

To purify ferritin, we followed Prof. Beck's instructions and used heat precipitation, IEC, and SEC methods. By using negative staining with TEM, we confirmed the assembly of the ferritin container.

Although we have not yet achieved the encapsulation of the components in our project, we plan to incorporate Prof. Beck's suggestions after the iGEM competition.

We express our gratitude to Prof. Dr. Tobias Beck for his interview and support. He showed great commitment and interest in our project and offered his ongoing assistance. His guidance was particularly beneficial in the early stages of the project and laid a crucial foundation for our progress.

Additionally, we appreciate his generous donation of the plasmid containing the wild type ferritin and the continued support by his PhD student Laurin Lang with our inquiries regarding the purification protocol.

With the design of our container, we were confronted with the problem that we have to get our delivery-system into the pathogens. That is when the idea of cell-penetrating peptides (CCPs) popped into our heads. CPPs have the potential to penetrate any cell membrane, even though the exact mechanism behind it is yet to be understood.

Dr. Dirk Becker is well known among us students as he is a teacher for some of our classes. He works at the Institute of Plant Science and Microbiology as a group leader for Molecular Plant Genetics and uses cell-penetrating peptides for his research.

Since Dr. Dirk Becker works with CPPs in his daily research, he was the perfect person to talk to. It must be noted, that Dirk Becker works with plant cells, so his expertise can not be directly transmitted to bacterial cells and the potential of CPPs for their membranes.

Most important takeaways

1. Are cell penetrating peptides a valid option to import our Ferritin to the target bacteria?

   One of the main questions was, if CPPs are actually capable of importing our ferritin complex into the bacterial cells. So we asked Dr. Becker, how many CPPs we would need to achieve that. He told us, that for a protein with the size of 160 kDA one CPP is sufficient, they even managed to transport a 180 kDA protein with one CPP. In general: the larger the protein, the harder it is to transport it over a membrane.

2. How to add CPPs to our Ferritin?

   He recommended us to not bind the CPPs covalently to the surface after expression, but to rather synthesize it to our Ferritin plasmid on a genetic level. That way, the CPPs are expressed together with the ferritin and will be on the surface of the Ferritin. Another option would be to incubate the peptides with our ferritin protein, and they would bind to the surface.

3. Can CPPs provide cell specificity?

   According to Dr. Becker, his results with protoplasts do not imply any cell specificity of CPPs. Although, it again must be mentioned that he works with plant cells and therefore could not tell us, if they perform differently when used on bacterial membranes.

What we implemented into our project

Dr. Becker mainly works with the CPPs R9 (BBa_K4669001), R12 (BBa_K4669002) and TAT (BBa_K1202006) in different variations and offered to provide us already extracted proteins as well as plasmids of these three CPPs. R9 and R12 are synthetic CPPs, TAT is a peptide used by the HI-Virus to penetrate human cells.

After the talk we discussed some of the input Dr. Becker gave us and came to the following conclusions:

1. In order to ensure that we have enough CPPs per ferritin container to transport it into a cell, we will fuse on CPP to every subunit of our ferritin. So in theory, every CPP will have to drag one 22 kDA heavy subunit, which should definitely be in the possible range.
2. We settled for the option to fuse the CPPs directly to our ferritin. The N-terminus of the ferritin faces outward. By fusing it on a genetic level we can control where the CPPs will be positioned. This brings also an advantage regarding standardization and controllability, which will be important aspects in the future as Dr. Grunwald mentioned.
3. Since Dr. Becker has no experience of the CPPs’ mode of action when it comes to bacteria, our task now was to investigate how the CPPs perform on bacteria.
4. Dr. Becker also told us that from his experience CPPs are not specific. So to provide specificity, we have to find another solution and talk to other experts.

We thank Dr. Dirk Becker very much for his knowledge, time and help and are grateful that he offered us to use his CPPs. Thank you very much!

First encounter

A big part of our project is circling around providing specificity for a targeted drug delivery. From the beginning on we knew we would have to find a solution to not only have an advantage over traditional systems like broad spectrum antibiotics, but to also ensure protection of our body’s cells and microbiome. This specificity could also help to increase the effectiveness of the transported substances. In the talk with Dr. Becker we found out that our available CPPs are no option for providing specificity.

After some talks within the team we came to the first idea of using antibodies for a specific targeting. To minimize the size of our construct, we quickly decided on trying to implement the smaller version of them: nanobodies. Life Science Nord connected us with Dr. Alejandro Rojas-Fernandez who is the scientific director of Berking Biotechnology Biotechnology, a company founded in Chile. Berking Biotechnology is producing nanobodies in alpacas, generating bacterial libraries for recombinant expression of these nanobodies for their usage in the field of cancer diagnostics and therapies.

We arranged a meeting with Dr. Rojas-Fernandez and talked about our project. We proposed our first idea of using bispecific nanobodies as mediators between our ferritin and the pathogenic target cells. We knew it would be essential to express and purify the nanobodies and the ferritin constructs separately. Not only to provide the factor of modulability, but also to give the ferritin enough space to fold correctly.

Most important takeaways

The first thing we talked about was the general procedure of nanobody production. The production pathway is pretty similar to the commonly known way of producing antibodies in mice: a (part of a) pathogen gets injected into camelids, and they produce the nanobodies. Then, blood samples are taken and the nanobodies are getting characterized. In total there are four steps of these immunizations. The time span starting with the first immunization and ending with finding a perfect nanobodies takes around 4-6 months.

Afterwards we talked about the idea of implementing nanobodies and started to develop an improved system.

Dr. Rojas-Fernandez said it was indeed a good idea to use nanobodies. But he quickly gave us the advice to rather use one simple nanobody instead of a bispecific one for particular reasons.

The main reason is the complexity. With a bispecific nanobody we would have one component more. Dr. Rojas-Fernandez said since we do not necessarily need the ferritin-binding nanobody, we should just leave it out. Some of the explanations why it is unpractical are here:

Another point that should not be negleted is the following: By using a system like a bispecific nanobody we would rely on non-covalent binding between the container and the nanobody. First we thought it could be useful to have less mass to transport into the pathogen. But Dr. Rojas-Fernandez quickly said we should be able to predict how many nanobodies are connected to the ferritin at any point of the application. Otherwise, it will be hard to control or predict any reactions in the body, and we would have a hard time to get the drug delivery system on the market.

He proposed to us two ideas of implementing the nanobody:

Our to-do list after this first meeting

1. By using nanobodies we will provide a very high specificity, where the applicant has to know what nanobody against which pathogen to use before using it. Therefore, the species of the infectious pathogen has to be known before the application. Dr. Rojas-Fernandez proposed to us, we could design a new rapid test device to be able to identify which pathogen the patient is infected with via a blood sample.
2. Dr. Rojas-Fernandez gave us the advice to not use a bispecific nanobody. He proposed to either use click-chemistry for coupling the nanobody to the surface of ferritin or to fuse the nanobody on a genetic level to the ferritin. Our task now was to do some research on that.

What we implemented into our project

1. The test device
   This year, we are focusing on the therapeutic approach and not on a diagnostic product. But Dr. Rojas-Fernandez is right: our system could be modified and applicated in many different situations. When focusing on different pathogens, we would have to know, what pathogen is there. Luckily for us, last year’s team from Hamburg, worked on another approach: developing a test device for fast detection of antibiotic resistances using a split ribozyme. Even though the team focused on detecting specific bacterial resistances, it has also developed a tool for the design of a similar system for other targets of detection. Also, after talking to Dr. Thomas Grunwald we found out that there are already some rapid tests in development that could be used.
2. Coupling the nanobody to our construct
   The most important thing for us was to find a definite solution for coupling the nanobody to our construct. We did research on both the approach of putting the nanobody between the CPP and the ferritin and linking it via click chemistry.

   And then it clicked - From bispecific nanobodies to click chemistry.

   For us, one of the most important things of our project is the factor of keeping our system modulable. Meaning - looking into our vision of producing transFERRITIN on a larger scale for public usage - there is as little effort needed as possible. Fusing the nanobody between the CPP and ferritin would take away a huge point of modulability since it happens on a genetic level rather than on a “puzzling” level.

   That is why we put more power into composing a solution using click chemistry and eventually found it. Read more about it here.

   We stayed in close contact with Dr. Rojas-Fernandez throughout the whole process. He was helping us with finding the perfect position for the Amber codon and was so kind to send us some plasmids containing different nanobodies for testing. For our proof of concept we started using a GFP nanobody, but we are planning on trying the other nanobodies after the competition. During that procedure we can always rely on some help by Dr. Rojas-Fernandez!

   Special thanks to Dr. Alejandro Rojas-Fernandez for always providing quick answers and always looking for new ideas, approaches and solutions!

Now, that we have decided on using click chemistry to couple the nanobody onto the surface of our ferritin container, we did some more research. After finding out that we could use non-canonical amino acids (ncAAs) to implement one of the two components within the backbone of our ferritin, we had a look into previous iGEM years to get some help. We quickly found last years’ winning team of the Foundational Advance track: Freiburg.

Last year they have been working on implementing ncAAs into compartment proteins and were happy to help us after we reached out to them. They taught us most of what we now know about introducing the Amber stop codon, co-transforming with a helper plasmid and what to do with the ncAAs.

In addition we were lucky: They continued to work on that part of their project afterwards and just released a new data bank for most of the known ncAAs and their properties called iNClusive. With the help of the great work of that team we were able to identify a fitting ncAA: TCO* Lysine. Read more about that here.

They also warned us about the significant decrease of the expression efficiency when working with that system. Due to the lack of time we were not able to do tests concerning that, let alone to optimize the expression, but we are planning on giving the optimization a shot after the Grand Jamboree.

Special thanks to Michael Spädt and Leon-Samuel Icking from last years’ iGEM team Freiburg for always being there to help!

With transFERRITIN we are developing a drug-delivery system meant for an application into the human body. So from the beginning on it has been a big matter to us to talk to an immunologist. With Dr. Clemens Wülfing we found an experienced one: he is the scientific head of a group on the field for interdisciplinary neurobiology and immunology at the University of Hamburg, and he is the head of research at the working group for interdisciplinary neurobiology and immunology - INI . We met him at the INI to discuss our approach with him.

We talked with him about the immunogenicity of the components of our construct. He gave us some input concerning the application and discussed possible alternatives with us.

Most important takeaways

1. General Guidelines Dr. Wülfing introduced us to two basic concepts:
   1. It is better to cooperate with the body instead of competing with it. Meaning, we should try to support the body’s immune system rather than bringing in new components that will stand in competition with the body’s immune response.
   2. The more synthetic our construct is, the more there may be a risk of an immune response against it.
2. Immunogenicity

   After presenting Dr. Wülfing our project we talked with him about our approach and asked him what to watch out for. Here we display some of the most relevant information he gave us:

   In general, he said, it is hard to predict whether and to what extent substances are immunogenic. It is likely that as soon as you bring in new substances or protein constructs into the human body - even though it might be a human protein just slightly changed - there will be an immune response.

   For our purpose it would be devastating to cause a strong response. Our system is supposed to help when the immune response is overwhelmed during an infection. To stress it even more would unnecessarily harm the body.

   Dr. Wülfing told us there are some limited ways to avoid causing an immune response:

   1. Suppress the immune system of the patient.
   2. Find a masking mechanism, similar to the ones, some viruses have.
   3. Create surfaces of proteins that are known to the body and will not be recognized as a threat.

   We dived deeper into a discussion about the last possibility of avoiding a response and how we can implement it into our project. More on that later.

3. Immunogenicity of CPPs

   The good news first: Dr. Wülfing said for one of our CPPs there is a high chance of it not being immunogenic: TAT. TAT peptides naturally occur in HI viruses. These viruses are known to be able to hide from the immune system, so we assume the CPP would not cause an immune response.

   The bad news: R9, R12 and all other synthetic cell penetrating peptides probably would cause an immune response.

4. Immunogenicity of Nanobodies

   The case for the nanobodies is most likely the same as for synthetic CPPs. But Dr. Wülfing gave us additional important input concerning the nanobodies:

   When introducing highly specific nanobodies that are unknown to the body, we would not only be likely to cause an immune response. If the pathogen, despite having a resistance or not, is already known to the body, the body will most likely have memory B-cells that would come into play when fighting it. The potential risk of blocking the binding sites of the antibodies produced by these B-cells would always exist. Therefore, we would not support the body’s defense mechanism but rather harm it. Hence the transFERRITIN approach would only be useful when it is the first time the body is infected with the pathogen.

5. Application

   Like we did with Dr. Grunwald we also discussed the way of applying transFERRITIN into the body with Dr. Clemens Wülfing. We talked about both the oral pathway and the infusion pathway via the blood. Basically both ways are possible, we just have to consider some points depending on the way we choose. Here is what we learned.

   1. Oral application

      When applicating transFERRITIN orally we could easily target pathogens in the intestine.

      We were worried that we would have a problem passing the stomach. The extremely low pH value could harm our system in terms of disassembling it. In fact Dr. Wülfing said that we do not have to be worried about that. There are several known ways of saving substances from the low pH-value, for example by packing it up into a shell thats stable even at extreme pH-values.

   2. Infusion into the blood

      When it comes to do an infusion into the blood to apply the protein container we would barely have problems with the application itself. The transFERRITIN complex, completely consisting of proteins, is highly water-soluble and could simple be stored in a solution.

6. Possible alternatives to avoid immunogenecity

   Now, that we learned that CPPs and the nanobodies could cause us problems, we discussed alternatives and Dr. Wülfing quickly came up with three of them:

   1. Defensins - an alternative for CPPs

      A first alternative that Wülfing mentioned are defensins which we could use instead of cell penetrating peptides. Defensins are small (30-40 amino acids long), amphipathic antimicrobial peptides that are part of the defense response in animals and plants. Their mode of action exists probably in penetrating the membrane with their hydrophobic region and therefore forming a pore, making the membrane permeable. Their selectivity is given by preferring membranes with a high cholersterin content. Thus, they mainly attack microbial membranes and spare eucaryotic membranes.

   2. Pattern Recognition Receptors (PRRs) - an alternative for nanobodies

      The second alternative Dr. Wülfing told us about are pattern recognition receptors (PRRs). PRRs are constitutively present on macrophages and dendritic to bind to pathogen-associated molecular patterns (PAMPs) that are presented by the pathogen. They are part of the innate immune system and therefor not immunogenic. Wülfing said we could technically use these receptors to build a new “nanobody”. These receptors are not as specific as nanobodies since they recognize patterns like mannose on the surface of pathogens. With that we would not have to rely on the diagnosis of the species of the germ before applying the medicine. We were thankful for that input and thought it was worth it to put some research into if this is a fitting alternative.

   3. Complement receptors - a second alternative for nanobodies

      The last alternative that came into the mind of Dr. Wülfing were the complement receptors. He said, we could take advantage of the immune system doing the first steps by marking pathogens with proteins of the complement system. These proteins then are displaying binding sites for complement receptors which we could fuse to our ferritin complex.

To-do-list after the talk

1. We had to do some research: Are defensins a valid alternative? And if so: which defensins would be a good substitute for CPPs? How can we fuse them to the ferritin?
2. The same goes for pathogen recognition receptors (PRRs). Can we use them? Or can we rely on complement receptors?
3. If we want to come closer to getting transFERRITIN on the market we will have to think about testing the immunogenecity.

What we integrated into our project

1. Sadly, we can not reliably test the immunogenicity of our system. We asked Dr. Wülfing if there is another way of testing it besides tests on animals. Unfortunately he said it is really hard to mimic the interactions between body and drug in a cell culture since only minimal parts of the body’s ways of reacting can be recreated there. So in the context of iGEM we can not test that aspect of our project. But what we can do is to minimize the risk of an immune response by searching for alternatives.
2. Defensins

   The talk with Dr. Wülfing came quite convenient for us since we were not as happy as we expected with the penetration results of our CPP tests. First, we did some research on if defensins could be a fitting alternative for us. We found out that defensins, due to their amphiphatic characteristic, are not really penetrating the cells in the sense of entering them and getting into the cytoplasm. They are more likely to settle down in the lipid bilayer of the membrane and not move out again. Due to that mechanism they form pores in the membrane and the cells are becoming leaky.

   We are not sure if that meets what we are looking for. With that system it is likely that the penetration by the defensins would kill the cell and not the substances encapsulated in our ferritin complex. But of course, we will have to test the behaviour of the defensins when fused to ferritin before completely removing them from our list of possibilities. Unfortunately we were not able to test them in the short period of iGEM. We are currently designing new sequences and will implement them in the next step of our testings.

3. Pattern Recognition Receptors (PRRs)

   As previously mentioned Dr. Wülfing introduced us to the idea of implementing PRRs instead of nanobodies. He already told us some features that would be in our favor: They are part of the body’s immune system and thus would most likely not cause an immune response. In addition, they are not as specific as nanobodies which would give us the ability to act without being dependent on diagnostics. But we had to put some more effort into doing research. Here is what we found out:

   PRRs, for example toll-like receptors (TLRs) are bigger than nanobodies. We will have to see if that affects the mode of action of transFERRITIN. They do not recognize anything cells of the human body express, so they will not target the body of the patient. But, they do not differentiate between good and bad bacteria. This could be a problem when applying transFERRITIN orally. As well as for the defensin we will have to test the extent of that change.

4. Complement receptors

   The last part are the complement receptors. As well as the PRRs, they are part of the innate immune system. But one of the main differences are the targets. While PRRs recognizes several bacterial patterns like mannose or lipopolysaccharides, complement receptors are only binding to other parts of the complement system. These parts are only placed on a bacterial membrane to mark it after the immune system recognized it as a threat. Thus, we are not only sparing the body’s cells, reducing cytotoxicity. We also would most likely protect the established microbiome. We are also planning to do some research on that.

Providing a modulable system it will be comparatively easy to change single components, so we really look forward to testing out so many new possibilities.

We want to thank Dr. Clemens Wülfing for quickly helping us, for finding joy in developing our project further and for establishing new principles we could use. We really had a fun time talking to you! Thank you!

When starting a project, it is essential to keep an eye on what is coming in the future in order to know how to properly shape the present.

In our case of designing transFERRITIN as a therapeutic tool, we have to consider its regulatory market approval. In order to do so, it is mandatory to do a lot of testing and to be evaluated at several steps of the process. Testing is expensive which is why scientific groups are often looking for help provided by big players of the pharmaceutical industry. One of these players is the company medac. This company, established in 1970, has over 2000 employees who evaluate, develop, and market clinical products in the fields of autoimmune diseases, oncology and urology. It is a privately owned company that has an eye on the patient’s wellbeing.

We talked to the head of the pharmaceutical and diagnostic development: Dr. Thomas Grunwald. He graduated with a master’s degree in chemistry and got his PhD in Hamburg before he worked with several different groups. Among them the Nobel prize winner Sir Gregory Winter. He also joined a start-up at one point of his career and completed a Master of Business Administration which gives him the ability to understand their client’s perspective on developing a new product.

For us, it was important to talk to an expert from the pharma industry to get input about what we could change already while our project is in its baby shoes to provide high standards even in the early phase.

Most important takeaways

After we had a nice talk about Dr. Grunwald’s career, we presented him our project and discussed some points of it.

1. About the whole system

   The first thing Dr. Grunwald said after the presentation was “Why so complex?”. The reason for this question was the following: The more different components we have, the more interactions between each of the components we have to study if we want to apply transFERRITIN to humans. And the more components the more tests concerning immunogenicity we have to do. In summary, you can say: The more components, the more quality assurance and money is needed. This will lead to a more expensive testing and production of our product.

   From the beginning on we planned to build up a modulable system of transFERRITIN. So, for us, it was quite shocking when Dr. Grunwald said that from a pharmaceutical perspective it is better or at least easier to have little modulability, because for every change of the system we would have to go through the process of regulatory approval again, which might include expensive clinical studies.

   Nevertheless, we went through every component of our project to discuss their qualification of using them in a therapeutic context.

2. About ferritin

   For the ferritin part of our project Dr. Grunwald brought up another interesting idea for the usage of it: he proposed we could load it with iron on purpose to cause oxidative stress on the target cells. Combining that technique with our approach, we could stress the pathogens with one more component besides the chemicals.

3. About the antimicrobial substances

   We then talked about what we want to encapsulate into our container. Here Grunwald told us we definitely have to find a way to prove that we get the same amount and composition of substances into the ferritin every time. It has to be standardized. Also, we have to prove the enclosed substances are stable in various environments and conditions that occur in the body in the course of the degradation process. And the big question is: how much can we transport with one ferritin molecule? Besides just calculating it we have to prove it whether there are differences in the calculations and reality. How much ferritin do we have to apply for a patient to have the effect we wish to have during an infection.

4. About the nanobodies

   The next component we discussed next is the nanobody. We already talked to Dr. Alejandro Rojas-Fernandez about the issue of our system possibly being too specific due to the nanobody. Rojas-Fernandez proposed to us to develop a first draft of a diagnostic device to identify the pathogen beforehand applying. Dr. Grunwald explained to us that it is not unusual to have a therapeutic product which depends on another diagnostic method. These are then called “companion diagnostics”. He said there is a high chance of a diagnostic device already being in development, and we should have a look into that. This should not be a burden to providing specificity.

   Dr. Grunwald mentioned providing specificity is a good and simple way to potentially increase the efficiency of a product. Of course, more specificity means more clinical studies needed to provide the targeting of as many pathogens as possible, and we should have a look into where to draw the line between specificity and coverage of multiple pathogens.

   One issue he sees not with using nanobodies but with the way we want to implement them into our system is concerning the click chemistry reaction. Right now, we can not say how many nanobodies will bind to the ferritin container. But what applies for the substances, also does apply for the nanobodies: we have to know how many of them are in our mixture at any point of the process. It is important that all processes can be standardized in terms of quality assurance, so that the same product can be produced with the same quality in every production step (keyword: standard operation procedures, SOPs).

5. About the CPPs

   Regarding the CPPs the only thing we should worry about according to Dr. Grunwald is that the CPPs could aggregate and hence block the syringe when applying or hence could interfere with the mode of action of transFERRITIN. But Dr. Dirk Becker said it would not be an issue. Of course, we have to prove it before we can launch transFERRITIN.

   Dr. Grunwald suggested that we consider using specific CPPs rather than nanobodies to make the complex simpler. However, whether there are any of the many existing CPPs that specifically target only one pathogen must first be verified. We also spoke with Dr. Clemens Wülfing about possible alternatives to nanobodies.

6. Which way of application to use

   Here is some general information about how a product has to be handled that Dr. Grunwald told us:

   1. Medicine should have a shelf life of at least two years, if possible.
   2. When producing proteins as medicine, they have to be cooled at every point of handling, including transport and storage in the clinics.
      1. That means temperature regulated trucks and fridge space in clinics is needed. If the cold chain is interrupted, you must not use the drug anymore.
      2. A way to avoid this could be to freeze dry the proteins and give away the powder which then can be often stored at room temperature and dissolved in a solution again.
      3. Hence, products that do not have to be cooled are easier to handle, and they are often more stable.
   3. We have to study the pharmacodynamics and pharmacokinetics of our product into every detail.
   
   

   Afterwards we talked about the way of applying transFERRITIN into the body via an infusion into the bloodstream. Here is what we learned:

   1. Easy application since proteins are soluble in aqueous solutions.
   2. The solution has to be absolutely sterile, and we have to be able to prove that and after which time span transFERRITIN leaves the bloodstream.
   3. It must be compatible with the blood serum of the patient.
   4. Aggregates must not be formed since it could block not only syringes but also the blood vessels.
   
   

   Dr. Grunwald said an application into the intestine via suppositories or gastric acid-resistant capsules is also possible, but we did not go into more detail.

7. About getting market approval

   When we really want to think about launching, we need to consider two main aspects, Dr. Grunwald said:

   When getting regulatory approval, a product always gets compared with other approaches, following the question, if there is an easier, less expensive, or more efficient approach. It is about the competitiveness and the risk-benefit ratio of therapeutics. As developers, we would need to find our competition and figure out what makes us unique.

   For example, one of our unique selling points is that we are developing a system that is preventing the emergence of new antibiotic resistances rather than running after them by developing new antibiotics. However, since new, effective antibiotics are often initially held back as reserve antibiotics, the investment of pharmaceutical companies in the development of new antibiotics is often not profitable. It therefore makes sense to find alternative approaches, as we have done with our system transFERRITIN.

   The second point Dr. Grunwald told us that in drug development we have to try to predict what will be needed in about 10 years. This is how long it can take for a product to reach the market. And in order for the product to be needed and marketable, you have to estimate what the future situation will be to avoid bringing obsolete technologies or drugs to market.

8. Presenting our project to investors

   Finally, Dr. Grunwald gave us some tips about how to present ourselves and our project when we want to find investors:

   First, we would have to display our business case: how many patients are there because of the illness we want to treat? How much does it cost society, not to have a better solution by now? How could society and economy benefit by using our product? How profitable is it for the investor?

   He also said we should definitely think about other ways we could use our system. Just in case the first idea is not working. He gave us some examples in which he reported that other companies, which are now successful, also had to adapt their ideas more often in the beginning, so that the product is useful for other purposes, and they had a need for it. But he absolutely sees potential in our project being transformed.

9. General principles that Dr. Grunwald told us that should always be kept in mind when developing a medicinal product:
   1. Keep it simple.
   2. Do not develop a problem for your solution but develop a solution for a real problem.
   3. We have to establish standardized methods for every single step of the manufacturing process.
   4. We also have to develop standard operation procedures (SOPs) for handling and applying the product after it is out of our hands later on.
   5. When starting the journey of bringing the project on the market, it does not have to be perfect. Often the product will undergo changes anyway, however try to plan as good as you can and accept that there are risks.

What we implemented into our project

For us, being an iGEM team that only had a year to develop something, we were only able to do the first, small steps for our project. Thus, we are limited in implementing advice that is looking so far in the future. Still, we took the time to discuss some points:

1. Test Device: Currently, there are already research groups working on the development of a diagnostic tool to identify more quickly the exact pathogen causing sepsis to be able to administer an effective antibiotic. For example, Prof. Dr. Ute Neugebauer is researching optical methods for sepsis diagnostics. An overview of current methods can be read here. Therefore, our specific application of transFERRITIN could cooperate with the future diagnostic test devices.
2. What we are now planning to test in order to come one step closer to becoming certified:
   1. Is our complex forming aggregates? How stable is the folding?
   2. To what extent is it soluble in aqueous solutions?
   3. How many nanobodies are binding? How much of the substance are we getting inside the ferritin container? Can we standardize it?
   4. Stability tests for every single component in different surrounding conditions.
3. Once we can produce all the components in a standardized way and the in vitro experiments work, we can test the pharmacodynamic and pharmacokinetic issues in subsequent animal studies.
4. Even though we are focusing on bacteria, we keep in mind that our transport system transFERRITIN can also prove useful in other areas for alternative approaches. Read more about that on our outlook page.

We want to thank Dr. Thomas Grunwald for quickly finding the time to come and visit us at the CSSB and for giving that much interesting and useful input. We were truly delighted listening to what you said, all coming from many years of experience. Thank you so much!

Jonas Ide is a venture capitalist, working for Evonik Venture Capital. Evonik itself is specialty chemicals company with an annual revenue of 18.5 billion Euros in 2022. The Corporate Venture Capital unit invests in promising start-ups around the world in fields, that Evonik themselves declared as growth areas, i.e. healthcare. Jonas’ responsibility is the financial field of an investment. He oversees the business plans and creates own models as well as sourcing investments in the area of healthcare. We talked a lot about how an interaction between Evonik Venture Capital and a potential start-up would enfold, how the contact is established, what Evonik Venture Capital offers its partners and what is a straight exclusion criteria for them. We also talked about investors in general and how to approach them and sell our idea. Afterwards, we introduced Jonas to our project and discussed our possibilities, our possible next steps, the potential of our idea and what we should do or avoid. We talked with Jonas about how we can continue with our project after the iGEM competition and how to become relevant for investors and successfully build a start-up with financial support from external partners.

Most important takeaways

• Our product is very modular and could be used in a wide field of applications. To be relevant for investors, we need to focus on the most promising area and develop a proof of concept to present to interested partners.
• Be clear about what you want to get from investors, how much you are willing to give away and how much the start-up is worth at any given time.
• Venture Capital companies use pitch events, conferences and other events to network and engage with potential partners. In order to have a high chance of being seen, attend these events, make yourself visible and sell your idea to the audience!
• When approaching a potential investor, make sure to know who you’re talking to and if your project even fits their portfolio.
• Don’t underestimate scaling. The process must be optimized, and you have to scale up potentially a number of times, not just once.
• Go out and sell! Build up relationships, attend events, network. The sales pitch should answer these three questions:
  • What problem are you solving?
  • Why is this important to your customer?
  • Why should they give you the money to solve it?
• Look for investors regarding the stage of your project. There are investors who fund very early phases of a start-up, but also some who only invest in the later stages.

To-Do list after the talk

• Build a diverse group of people with a variety of skill sets to be prepared as good as possible for the challenges of building your own start-up.
• Do an extensive market research and ensure our product is needed and wanted!
• Create a presentation about our project that not only covers the theoretical background, but also sells the product to the relevant audience and potential investors. Make sure to present the problem to the audience and why your approach is the best way to solve this problem.
• Make sure we have a working proof of concept, so we can present and validate our approach.
• Develop a business plan on how we approach the next phases of our project. State what needs to be done to commercialize our product.
• Sort out the situation regarding your IP (intellectual property) with our University. It must be clear, which knowledge belongs to whom.
• Take part in a start-up event or something similar to build on our project concept.

What we integrated into our project

• We created a presentation that fits the criteria listed above to approach potential investors.
• We work on our proof of concept to penetrate E. coli with specific nanobodies and release our cargo. Afterwards, we want to effectively target and penetrate P. aeruginosa as our main application.
• We worked out a business plan we can follow after the iGEM competition. You can take a look at it under Entrepreneurship.
• As a team that participates in the iGEM competition, we were interdisciplinary from the get go. Still, our team must be further diversified and committed to the project in order to proceed with our idea and realize it.

Organizations like the World Health Organization (WHO) and the media often emphasize the need for alternatives to antibiotics, citing statistics such as „more than 2.8 million drug-resistant infections each year and more than 35,000 people die as a result.“ These numbers may instill fear but can feel distant. With these numbers, the average person must have had contact with individuals who suffered a serious infection.

For our project we want to get to know this person and after speaking with various people, listening closely and recalling past conversations, we found some people who had experienced a serious infection themselves or have had close friends that have. One of these individuals is Prof. Dr. Carsten Lübke, a Professor of cell biology and tissue engineering at the Berlin University for applied sciences.

After reaching out to him, Prof. Dr. Lübke graciously agreed to share his story.

The Story:

It all began in the late 1990s or early 2000s when he developed a mild cold during the winter months, seemingly nothing to worry about. When common remedies failed to alleviate his symptoms, he decided to consult a doctor. After a brief visit, the doctor prescribed antibiotics, which he took as directed, typically over a span of seven to ten days. However, after the treatment Prof. Dr. Lübke remained ill. His condition led him back to the doctor, who conducted diagnostics of the lung that initially showed no concerns. Yet the doctor prescribed new antibiotics, without apparent concern. As before, the medication had no effect. In contrast, his condition deteriorated, and he developed a fever of around 40 °C. With no improvement, worry began to set in.

At the time, Prof. Dr. Lübke was working as a scientist at the University Clinic, Charité, in Berlin. He was well aware of the term “antibiotic resistance”, so he admitted himself to the Charité. The Charité took his infection seriously and immediately assigned him to an isolation unit, due to the ineffectiveness of two different antibiotics, of which one was a broad-spectrum antibiotic. The results of the previous diagnostics of the lungs were reviewed and showed indications of an early infection. This led to the repetition thereof revealing clear signs of an infection. Samples were taken immediately for identification of the pathogen.

The diagnosis: an atypical lung infection caused by Mycobacteria

With the diagnosis and unsuccessful attempts at antibiotic treatment, there was no choice but to rely on his immune system. Fortunately, he was offered participation in a clinical study for a new treatment. As a scientist, he understood the importance of such studies for research, and as a patient with a resistant infection, he accepted the risk and the offer. He underwent a painfully burning infusion with unknown content, which, after about a week, finally relieved his symptoms. Apart from the initial infusion, he experienced no problems with the medication and successfully overcame the infection. Nevertheless, his body remained weak and simple movements felt like an endeavor. It took approximately two months for him to fully recover and return to his former self.

Reflecting on his experience, Prof. Dr. Lübke questioned why his doctor hadn't referred him to the clinic proactively or why a second antibiotic had been prescribed despite the ineffectiveness of the first. Given the context of antibiotic resistance, this should have been a clear warning, particularly for general practitioners.

In hindsight, he suspected that he may have contracted the infection while working with human samples in the lab. It was common for him to receive tissue samples and process these in the lab. It might have been possible that a pathogen entered his system on an aerosol while cleaning the lab equipment.
This experience heightened his awareness, not only for himself but also for those close to him that antibiotic resistance is a serious threat that needs to be solved before infections are untreatable.

Most important takeaways

1. The prescription of Antibiotics

   During the late 1990s and early 2000s, antibiotic resistance was already a known concern in healthcare. However, not all doctors were fully aware of or took this issue seriously.

   Prof. Dr. Lübke's doctor prescribed antibiotics prematurely and followed up with another prescription for a different antibiotic without providing information about the possibility of antibiotic resistance or the risks associated with improper usage.

   This problem appears to have persisted even in 2023, with some doctors still prescribing antibiotics imprudently.

   While there has been some progress, antibiotics are typically prescribed only when visual signs of bacterial infections are evident, accompanied by clear instructions on correct usage. Nevertheless, information on antibiotic resistance or alternatives methods for treatment is often lacking.

2. Mycobacteria

   Infections with mycobacteria usually leads to diseases such as Tuberculosis or leprosy, whereas some lead to pulmonary infections caused by nontuberculous mycobacteria (NTMs).

   After some research, we discovered NTMs have proven to be an emerging threat. Mycobacteria are challenging to diagnose due to their slow growth, and they possess both intrinsic and acquired resistance against antibiotics, which makes treatment laborious.

   Their ability to produce biofilm and their thick cell contribute to their intrinsic resistance, while acquired resistance has evolved by prolonged antibiotic treatments as well as their high ability to acquire protective mutations during single drug treatments. (Saxena et al. (2021))

   Pathogens like the NTMs highlight the need increased awareness of antibiotic resistance and the urgency for novel diagnostics and treatment options.

3. Alternatives to antibiotics

   Before Prof. Dr. Lübkes hospitalization, alternatives to antibiotics were not known to, or given to him. However, research on methods such as phage therapy, which utilizes bacteriophages - viruses specialized to bacteria, was already underway. Despite its potential, current EU regulations limit the use of this therapy to specific cases.

   Other research areas include vaccines, immunotherapheutics, CRISPR/Cas-editors, probiotics, fecal transplant processing, antimicrobial proteins and Nanobiotics & Enzybiotics. Additionally, the search for novel antibiotics continues, although their long term potential remains uncertain (Kumar et al. (2021))

   Another possible alternative to antibiotics is our project - transFERRITIN a modular drug-delivery-system based on ferritin.

   Prof. Dr. Lübke responded very positively to our project, emphasizing its creative, thoughtful and thrilling it is. He noted that antibiotic resistance is one of the most significant challenges that the life sciences community must address, and he's pleased to see young students taking on the challenge with exciting and creative solutions.

To-do-list after the talk

1. Following our conversation with Prof. Dr. Carsten Lübke as well as chats with friends and family, it became clear to us that the concept of antibiotic resistance is not yet completely understood by the general public. We sought to assess the knowledge on this issue.

What we integrated into our project

1. To get the bigger picture, our public engagement division conducted a survey on antibiotics and antibiotic resistance. Click here to read more about it.
2. Early on, our team had the assumption that the general public has a misconception of synthetic biology and antibiotic usage. Our public engagement division proactively took this matter into their hands and conducted a range of activity’s.

   These include:

   1. iGEM@School
   2. Girls Day
   3. Day of Families
   4. Career Orientation Day
   5. German Reunification Day (TDE)
   6. Games designed by our Team

   These are only some examples, if you follow this link to our public engagement site you will find more

We express our thanks to Prof. Dr. Carsten Lübke for his time and willingness to share his story. We truly appreciate it since a story like this is not always easy to share. His candor made this conversation delightful, thank you!

Last but not least we want to mention the survey we did. When talking about finding a solution for a public issue we have to know to what level that public is involved in the issue. Only when gaining that knowledge it is possible to assess the importance of certain parts of the project.

In that survey we asked questions about experience and knowledge when it comes to using antibiotics and being aware of the rising crisis of emerging antibiotic resistances.

Even though it did not have an impact on our project outline or lab work it still had an effect on how we saw the issue we are addressing and preparing fitting, solution oriented projects for Public Engagement. Read more about the survey and its results here.

After getting the results of the survey we figured there is still the need to inform the public more about the danger of antibiotic resistance. Find out more about what we did to spread awareness on our education page.

When looking for an expert on the field to talk to it can be hard to find the best fitting one. That is why we reached out to Life Science Nord (LSN). LSN is an organisation that connects over 500 companies, universities and research institutions in the field of biotechnology and pharmacy in Hamburg and Schleswig-Holstein in Germany. Throughout the years they built up a huge network of partners and contribute to the process of innovation and value creation of northern Germany. They already were a great help for last years’ iGEM team Hamburg. That is why we reached out to them again.

After having a great talk about the idea of our project they did not hesitate to forward us to some of their partners. That is how we came to our amazing talks with Dr. Alejandro Rojas-Fernandez, Prof. Dr. Johannes K.-M. Knobloch and Dr. Thomas Grunwald.

Thanks to Life Science Nord and especially Georg Eschenburg for taking the time to help us with our project! This contact really boosted us forward. Thank you!