Human Practices

Key Implementations

• Tested Sushi S1 activity at different localizations by fusing the sequence to different signal peptides.
• Adapted protocols for killing assays with synthesized Sushi S1 peptide.
• Used a constitutive promoter for eCPX expression in OMV producing bacterial strains, for a homogenous population of OMVs.

Purpose
As one of our two Principal Investigators Dr. Nicole Gensch was an indispensable help during the whole project. She was involved in every step of CAPTURE and supported all subgroups equally with her knowledge, experience, enthusiasm and encouragement.

Contribution
Dr. Gensch was involved in the project in every aspect inside and outside the lab. She not only helped with questions regarding our experimental work but also with coordinating the whole project and team. She was the first point of contact to get another perspective on any matter.
With her extensive knowledge on synthetic and microbiology, Dr. Gensch actively pushed our project forward with numerous ideas for improvements and further experiments for all subgroups. Additionally, she ordered all materials and substances needed for our experiments and generously donated multiple resources like plasmids or enzymes from her own lab.
She introduced various team members to the handling of mammalian cell cultures and laboratory devices like the autoclave. Already before starting lab work, Dr. Gensch gave the whole team an introduction on how to work safely in a biosafety level 1 laboratory.

Implementation
To give some examples of her multiple contributions to the success of the project, we listed the most impactful below.
One of the most impactful suggestions that were implemented into our project was the analysis of the effectiveness of our AMP Sushi S1 at different cellular localizations. This idea was additionally supported by Prof. Dr. Burkhard Tümmler. Their proposal initiated the design and testing of three different signal peptides to investigate how our AMPs exert the biggest effect on bacterial growth. This way we could identify that the secreted versions of Sushi S1 and Conga impaired E. coli growth rate the most.
Regarding the analysis of the effect of synthesized AMPs, Dr. Gensch contributed significantly to the protocols of our killing assays with synthesized peptides, suggesting to test the effect in various media to minimize possible interference between peptides and environment.
She was also instrumental in introducing a constitutive promoter for eCPX-SpyTag expression used within OMV functionalization for enhancing eCPX production.
Furthermore, Dr. Gensch proposed to not only test the concept of CAPTURE in our project’s model organism Pseudomonas fluorescens but also in the standard organism E. coli which lead to the design of plasmids to test Sushi S1 and Conga expression in E. coli BL21 (DE3).

Key Implementation

• Improved the western blot protocol to successfully show Sushi S1 expression after induction with IPTG.
• Suggest the use of BCA assay to estimate OMV concentration.

Purpose
Dr. Pavel Salavei is one of our two Principal Investigators (PI) and contributed significantly to all steps throughout the project. All subgroups benefited from his knowledge and experience, his constant feedback, input and his support regarding almost every decision from start to finish.

Contribution
As an expert on protein expression and purification, Dr. Salavei was an indispensable help for all kinds of questions regarding SDS gels, Western Blots, expression conditions and protein purification. He was always available to improve protocols, find new methods or donate desperately needed materials to help advance our project. Especially to the Plasmid and OMV subgroups his expertise on the topic was most valuable and helpful.
Additionally, he was able to give an introduction to almost all devices used by our team throughout the project. As a member of the CIBSS Toolbox, Dr. Salavei introduced individual team members to work with multiple devices from plate reader, Fluorescent Activated Cell Sorting (FACS) to Dynamic Light Scattering (DLS). This was particularly beneficial for measurements regarding the Lipid-based Nanocarriers.
His constant efforts to help the team in every way possible by actively participating in all team meetings, giving his thoughts on every topic were highly valued and appreciated by the whole team.

Implementation
To name just a few of the many contributions that helped drive the project forward, some of the most important implementations are listed in the following.
One of the biggest challenges for the Plasmid subgroup turned out to be the proof of Sushi S1 expression in E. coli cultures after induction with IPTG. After multiple suggestions for adjustments to our protocol for sample concentration, cell lysis and SDS gel preparation by Dr. Salavei, we were eventually able to successfully detect the AMP Sushi S1 on our Western Blots.
For the work with Lipid based Nanocarriers the introduction to the DLS device was a necessary step to measure the size of the LNPs and extruded liposomes generated in our lab. Confirming the particles are in the right range of size was inevitable for downstream applications in fusion assays and the potential delivery into the lung. As the size of the particles usually appeared to be just below the resolution limit of our casual microscopes, with an average diameter in the range of 100 - 400 nm, establishing the DLS was essential to prove the presence of our carrier systems and continue further assessments with that knowledge as a basis.
Furthermore, he crucially contributed to the progress of the OMV subgroup by suggesting to use BCA assays to determine the protein concentration of OMVs. This allowed us not only to distribute similar amounts of OMVs on SDS gels and Western Blots but could also be used to estimate the overall concentration of OMVs.

Key Takeaway

• Test the functionality of synthesized promising antimicrobial peptide D-CONGA-Q7 in comparison to our initial AMP Sushi S1.
• Switch the peptide sequence in the plasmid; compare the effect of expressed L-CONGA-Q7 with synthesized D-CONGA-Q7.

Purpose
In our project, we are utilizing the membrane-permeabilizing ability of antimicrobial peptides (AMPs) to target P. aeruginosa in the lungs of patients suffering from pneumonia. Prof. Wimley, from the Tulane University School of Medicine in New Orleans, USA, is at the forefront of developing synthetically engineered AMPs as alternatives to conventional antibiotics, particularly against ESKAPE pathogens. In his recent work published in ACS Infectious Diseases (Ghimire et al., 2023), Prof. Wimley has demonstrated how AMPs, which often face challenges such as low solubility, can be synthetically evolved into more potent versions. Following his talk at our institute on his newest and most effective AMP, D-CONGA-Q7, we seized the opportunity to obtain his expertise in the field of AMPs.

Contribution
During his presentation, Prof. Wimley strongly emphasized the urgency of finding new ways to combat the growing global threat of antimicrobial resistance. Although AMPs are a promising addition to conventional antibiotic treatments, their use is often limited by high production costs, susceptibility to proteases, and low solubility. While Prof. Wimley is focused on improving parent AMPs through synthetic modifications, he was also very intrigued by our idea to produce the AMP directly within the pathogen we aim to target.

The main topic during our meeting was the stability of the AMP once produced in the pathogen. Due to their relatively short half-life, the peptide is degraded quickly after bacterial cell death. Although this should decrease the risk of a strong immune response in the lung, Prof. Wimley raised the concern that the bacterial cell might only produce enough AMP to harm itself. Therefore, only one bacterium per plasmid delivery would be killed. If we could implement some kind of delay mechanism, we could theoretically reduce the number of transport vesicles and plasmids during the treatment and also target bacteria that are able to develop resistance against the targeting systems on our carriers. He suggested that instead of encoding only one AMP on the delivered plasmid, we could include a string of AMPs interrupted by pathogen-specific protease cleavage sites. This approach would activate AMP functionality only after proteolytic cleavage, producing a larger volume of AMPs for each successful plasmid delivery.
In Wimley’s most recent work, the peptide D-CONGA-Q7 demonstrated a very high killing rate, especially against the ESKAPE pathogen P. aeruginosa. However, this version of the peptide consists of D-amino acids, decreasing susceptibility to proteolytic degradation. Since CAPTURE is characterized by the delivery of a plasmid that forces the pathogen itself to produce the AMP, using D-amino acids is not feasible. According to Prof. Wimley, peptides retain their functionality if for all amino acids the other enantiomer is used. Although he has not tested the L-configuration of the D-CONGA-Q7 peptide, he believes there is a high likelihood that the bacterially produced peptide would maintain its function.

Implementation
In the initial planning phase of our project, we decided to use a different AMP called Sushi S1, with the intention of comparing the effectiveness of various AMPs in our experimental setup. Prof. Wimley kindly offered us a small sample of his synthesized peptide, D-CONGA-Q7, to test its function in comparison to Sushi S1. Since our first experiments indicated that D-CONGA-Q7 was more effective than the synthesized Sushi S1, we also switched the peptide sequence in our plasmid. This allowed us to test whether the expressed L-enantiomer of the AMP is as potent as the synthesized D-version.

Outlook
Due to time constraints, we have decided to stick to our initial approach of encoding only one AMP on our delivered plasmid and postpone the implementation of Prof. Wimley’s suggested delay mechanism. However, we have already started researching Pseudomonas-specific proteases and their cleavage sites, in case we have enough time to compare the efficiency of the different strategies.

Key Implementation

• Used cationic DOTAP lipids for enhancing encapsulation efficiency.
• Established pre-staining DNA with DAPI before encapsulation in liposomes to visualize encapsulation under the microscope.
• To visualize the fusion experiment, we incorporated fluorescent DOPE-Atto 647N lipids.

Purpose
Prof. Römer is University Professor for Signaling Processes and Synthetic Biology at the Albert-Ludwigs-University in Freiburg. His research focuses on biological signaling processes, host-pathogen interactions and artificial cells.
As we are still in the early stage of our project, we aim to determine the best methods for producing liposomes. We are particularly interested in:

• Available liposome formation techniques and the time requirements for each method.
• Strategies for making liposomes specific for fusion with P. aeruginosa.
• Optimal types of lipids to use.

Contribution
Optimal Liposome Size
Prof. Römer advised that suitable liposome sizes range from 50 nm to a maximum of 100 nm, with an upper limit of 400 nm to avoid potential blood vessel blockages.
He outlined two primary methods for reducing liposome size:

• Extrusion: This mechanical process involves pressing pre-formed liposomes through a porous membrane, resulting in smaller vesicles.
• Sonication: This technique uses specific ultrasound frequencies to decrease liposome size.

He noted that FACS analysis might be challenging due to the small size of our liposomes.

Suitable Lipid Composition and Modifications
For fusion experiments, Prof. Roemer strongly recommended the use of cationic lipids. He also suggested incorporating fluorescent lipids for enhanced detection of fusion events and to improve imaging quality in microscopy. To assess fusion efficiency between bacteria and liposomes, Prof. Roemer advised adding liposomes to bacterial cultures on agar or in liquid media.
For lipid modification, Prof. Roemer suggested:

• Activatable lipids with N-hydroxysuccinimide groups for covalent binding to lectins.
• Biotinylated lipids as an alternative, using streptavidin as linker to lectins.
• Lectins should comprise 5-10% of the lipid composition, depending on fusogenicity.

DNA Uptake
We also wanted to know how we can measure the uptake of DNA in the liposomes. Prof. recommended staining DNA with DAPI or Propidium Iodide. However, according to Prof. Roemer, we have to consider the resolution limit of the microscope. With an excitation wavelength of 400 nm, the resolution is 200 nm (1⁄2 λ). If our liposome is only 50 nm in size, then we see a spot in the microscope image. Pre-staining DNA before liposome encapsulation could be potentially used for indirect visualization.

Outlook
For further expertise, Prof. Roemer suggested contacting:

• Prof. Alexander Titz for biofilm-specific questions.
• Prof. Jens Timmer for modeling assistance.
• IMTEK or Hans Schickard in Freiburg to find out more about nebulizer technology.

Prof. Römer generously offered access to his lab for experiments with P. aeruginosa under appropriate supervision.

2. Meeting 08.08.2024

Purpose
The purpose of the meeting was to address ongoing challenges with our lipid ratio and plasmid encapsulation when using the PVA swelling method. Additionally, we aimed to discuss and plan a joint fusion experiment with P. aeruginosa.

Contribution
Prof. Römer contributed valuable insights, suggesting we use DOTAP in a range of 10-30 mol% to trigger fusion with the bacterial membrane. It was noted that lower concentrations of DOTAP would stabilize the liposome, while higher concentrations would enhance fusion. We also discussed the electrostatic effects of DNA charge on the fusion process and encapsulation efficiency.
For the fusion experiment, Prof. Römer provided fluorescent lipids (DOPE-Atto647) and Gb3 lipids, which are crucial for the specific targeting of P. aeruginosa.

Implementation
We will integrate DOTAP at the recommended ratios in our liposome formulations. Additionally, fluorescent DOPE-Atto647 and Gb3 will be incorporated into our fusion experiment to ensure accurate targeting and tracking of liposomes.

Outlook
We had initially considered using an ampicillin resistance plasmid as proof of successful fusion, but Prof. Römer highlighted a major concern. Since fusion may only penetrate the outer membrane of gram-negative bacteria, the plasmid might remain in the periplasm and not be expressed. As an alternative, he proposed using fluorescent DOPC-Alexa 647 (0.5 mol%) in the lipid solution to trace fusion more effectively.

Key Implementation

• Start establishing a workflow in our lab that would allow the implementation of two methods, extrusion and thin film hydration, for downsizing our Lipid-based Nanocarriers.

Purpose
After implementing the first methods to produce lipid-based nanocarriers, GUVs respectively, we were faced with the issue that they were too large to effectively fuse with our target bacteria. Therefore, we were on the lookout for downsizing options. In literature extrusion was named as a common method to downsize GUVs, and to achieve a more homogenous size distribution. To establish this method in our workflow we contacted Dr. Pablo Rios-Munoz, who kindly introduced us to the procedure.

Contribution
Dr. Rios-Munoz gave us a detailed introduction, not only to the usage of the extruder, but also to the complete workflow of handling lipids, forming multilamellar vesicles (MLVs) with thin film hydration, downsizing them with the extruder and determining the successful formation and size of the resulting particles using the DLS. We learned that lipids are best stored protected from light and in the freezer. They are usually delivered as a powder which is then dissolved in a suitable solvent, in our case chloroform, and kept in air tight glass vials under a protective atmosphere using nitrogen or argon. When using chloroform as a solvent lipids are best handled on ice and pipetted with glass syringes. Syringes must be cleaned thoroughly with chloroform when switching the lipid composition. To form liposomes, the chloroform around the lipids is evaporated under an argon/nitrogen stream in a round bottomed flask, until only a thin layer of lipids remains at the bottom of the flask. Residue chloroform is evaporated in a vacuum. After achieving this, work should be continued above the highest phase transition temperature in the different lipids used. First the thin film of lipids is rehydrated in the desired buffer in a water bath, resulting in multilamellar vesicles forming. Once all the lipids are dissolved usually the MLV solution is freeze-thawed to be prepared for extrusion. Next the extruder is assembled on a heating plate, the desired filter is applied and the syringes are loaded with the MLV solution. After pushing the solution back and forth through the filter an uneven number of times, the solution can be discarded to a new vial. This is the final product, which can be measured with DLS to determine the average particle size, which correlates with the pore size of the filter used. After introducing us to the extruder, Dr. Rios-Munoz kindly agreed to lend us the device for the time of the project, along with providing us filters in different sizes.

Implementation
The advice Dr. Rios-Munoz gave us on handling the lipids was important in all further proceedings to create our delivery system. We were mostly interested in the extrusion, as our lipid-based nanocarrier formation method at that time was preferably PVA swelling resulting in giant unilamellar vesicles (GUVs), which we wanted to size down after proving the plasmid encapsulation in a microscope. However, learning about the thin film hydration method we considered this as an alternative method and began establishing a workflow in our lab allowing the implementation of both methods, being based on the same principle of rehydrating a thin layer of lipids. We adapted the extrusion protocol for the phase transition temperatures of our lipid composition and measured the extruded particles in the DLS after different times passing the filter to reduce sample loss by determining the least amount necessary to achieve our desired size distribution.

Outlook
We continued using extrusion as a standard method to control the particle size. However, we were also faced with the issues resulting from active downsizing of the lipids. Lipid-based nanocarriers might get destroyed during the process, releasing the encapsulated plasmid. It also poses a risk for contaminations of the sample with the extra steps of using syringes and filters. The resulting particles are only accessible in the DLS as they are too small for the microscope. This posed the issue for new staining methods and assays to prove the presence of plasmid and lipids and also accessing if the plasmid is encapsulated or not. We thus considered FACS and plate reader measurements to further characterize the resulting particles.

Key Takeaway

• Use PVA swelling as method to produce giant unilamellar vesicles (GUVs) allowing us to visualize these with confocal microscopy.
• Incorporate Gb3 as targeting ligand in our fusion experiments.

Purpose
Dr. Olga Makshakova is part of the lab group of Prof. Römer. She specialized in the production of Liposomes and Lipid Membranes and is trained to handle Pseudomonas aeruginosa in the infection lab.

Contribution
Dr. Makshakova played a significant role at the outset of our project by teaching us how to form liposomes using the PVA swelling method, a technique chosen for its efficiency in producing giant unilamellar vesicles (GUVs). She guided us through each step, explaining how the swelling of polyvinyl alcohol (PVA) films supports the formation of vesicles. Later, Dr. Makshakova demonstrated the analysis of our vesicles under the confocal microscope, where we were able to visualize the structural integrity of the liposomes. This analysis was crucial for verifying the liposome formation process and ensuring that the vesicles were suitable for upcoming fusion experiments. During one of our discussions, she shared valuable insights into the targeting mechanisms of Gb3. This lipid, known for its role in cellular recognition and binding, was particularly important for our project, as it provided a mechanism for the vesicles to interact with specific bacterial receptors. This targeting capability is essential for ensuring the successful delivery of the vesicles to P. aeruginosa.

At the end of the project, Prof. Römer, Dr. Makshakova and Dr. Serap Guinot enabled us to attempt the fusion of our vesicles with Pseudomonas aeruginosa, which requires high biosafety precautions due to its pathogenic nature. Because of these biosafety concerns, she personally conducted the experiment on our behalf. Afterward, we sat together at the confocal microscope, where we analyzed the potential fusion between the vesicles and the bacteria. This step was the finishing touch of our project, as it provided the first visual confirmation of the interaction between our lipid-based nanocarriers and P. aeruginosa.

Implementation
Throughout the project, we consistently utilized the PVA swelling method for liposome formation, refining and optimizing the technique as we progressed. After the initial confocal microscopy session with Dr. Makshakova, we knew it was possible to visualize and characterize the vesicles, ensuring the method’s reliability for future experiments.

Outlook
After the first meeting, we confidently adopted PVA swelling as our core method. For the fusion experiments, we incorporated Gb3 to target P. aeruginosa, demonstrating its potential for specific bacterial targeting. Future work will focus on optimizing Gb3-mediated fusion and exploring broader applications in targeted delivery.

Key Implementation

• To ensure a high encapsulation efficiency and avoid damaging mammalian cells, we utilized a lipid combination of DOTAP and the neutral lipid DOPE.
• Employed the extrusion method for downsizing giant unilamellar vesicles (GUVs).

Purpose
Anna Ruppl is a PhD student in the group of Dr. Allmendinger. She deals with the stabilization and manufacturing of mRNA-lipid nanoparticles by lyophilization. We met her together with Valentin Bender, as both are part of the Department of Pharmaceutical Technology and Biopharmacy at the University of Freiburg.

We are particularly interested in whether our liposomes remain stable after synthesizing and bonding with the plasmid. We also want to know whether direct transport through the airways is effective and feasible. In addition, both Ruppl and Bender are familiar with liposomes. We would like to seek their advice on which lipids are best suited for liposomes and how best to produce them and reduce their size if necessary.

Contribution
Ruppl told us that there are additional problems with the transport of mRNA in liposomes. We should therefore focus on achieving the highest possible fusion efficiency, as transport through liposomes should not usually cause lasting damage to the plasmid. They also advised us to use positively charged lipids, such as DOTAP, as these are more efficient for encapsulation and targeting than uncharged lipids. However, they emphasized that cationic lipids are more toxic to mammalian cells than uncharged lipids. The formation of liposomes should also be more difficult with cationic lipids. The surface modifications already presented by Römer, such as Gb3, can help us here. They also recommend that we read other papers that specialize in exact and specific formation. They recommend adding the plasmid during rehydration and PVA swelling. In general, they encourage us to test different options to create a comparison and draw our own conclusions. With plasmid encapsulation, they leave all options open, although they favor the former. To reduce the size of our GUVs, they recommend hand extrusion with Hamilton syringes and sterile filters. The lipid solution is pressed 21 times between the 2 syringes.
When we proposed our idea to administer the drug as an aerosol using a nebulizer (which we had already received positive feedback for from Dr. Römer, Dr. Tümmler, and Dr. Rieg), Ruppl and Bender suggested looking into the start-up RNHale, which is developing inhalable, RNA-containing lipid nanoparticles. However, they cautioned that this application is quite complicated. There are many parameters to consider, which would massively prolong our project. As an alternative, they suggested administering CAPTURE intravenously. From the meeting with Rieg, we learned that CAPTURE will passively attach to the lungs. So it remains a realistic alternative.
Mathematical modeling can be combined well with dual centrifugation, but they warned us that such modeling requires high computing power.

Implementation
For our next experiments, we plan to use the cationic lipid DOTAP, but we will probably use this lipid in combination with a neutral lipid, DOPE, to ensure encapsulation as efficiently as possible while trying not to damage mammalian cells. We are planning experiments to test this approach. We will also integrate our plasmids during rehydration and try to encapsulate our plasmids in liposomes for the first time. We will also try extrusion, as it seems to be the most effective method for downsizing liposomes after Bender, Ruppl, and Römer have explained it to us. We are getting support from the Signalhaus in Freiburg, where our laboratory is based.

Outlook
We are considering the possibility of using Gb3 to modify our liposome surface. We want to read more literature for future applications. As we are not planning to manufacture hardware, we just want to find out about a suitable application. Based on the information from Bender and Ruppl that ingestion with a nebulizer is rather difficult to implement, we definitely have to continue looking at intravenous and inhalation applications.

Key Implementation

• To ensure a high encapsulation efficiency and avoid damaging mammalian cells, we utilized a lipid combination of DOTAP and the neutral lipid DOPE.
• Employed the extrusion method for downsizing giant unilamellar vesicles (GUVs).

Purpose
Valentin Bender is a PhD student in the group of Prof. Dr. Suess. He specializes in RNA formulations for cardiovascular diseases. We met him together with Anna Ruppl, as both are part of the Department of Pharmaceutical Technology and Biopharmacy at the University of Freiburg.

We are particularly interested in whether our liposomes remain stable after synthesizing and bonding with the plasmid. We also want to know whether direct transport through the airways is effective and feasible. In addition, both Ruppl and Bender are familiar with liposomes. We would like to seek their advice on which lipids are best suited for liposomes and how best to produce them and reduce their size if necessary.

Contribution
Ruppl told us that there are additional problems with the transport of mRNA in liposomes. We should therefore focus on achieving the highest possible fusion efficiency, as transport through liposomes should not usually cause lasting damage to the plasmid. They also advised us to use positively charged lipids, such as DOTAP, as these are more efficient for encapsulation and targeting than uncharged lipids. However, they emphasized that cationic lipids are more toxic to mammalian cells than uncharged lipids. The formation of liposomes should also be more difficult with cationic lipids. The surface modifications already presented by Prof. Römer, such as Gb3, can help us here. They also recommend that we read other papers that specialize in exact and specific formation. They suggest adding the plasmid during rehydration and PVA swelling. In general, they encourage us to test different options to create a comparison and draw our own conclusions. With plasmid encapsulation, they leave all options open, although they favor the former. To reduce the size of our GUVs, they recommend hand extrusion with Hamilton syringes and sterile filters. The lipid solution is pressed 21 times between the 2 syringes.
When we proposed our idea to administer the drug as an aerosol using a nebulizer (which we had already received positive feedback for from Dr. Römer, Dr. Tümmler, and Dr. Rieg), Ruppl and Bender suggested looking into the start-up RNHale, which is developing inhalable, RNA-containing lipid nanoparticles. However, they cautioned that this application is quite complicated. There are many parameters to consider, which would massively prolong our project. As an alternative, they suggested administering CAPTURE intravenously. From the meeting with Rieg, we learned that CAPTURE will passively attach to the lungs. So it remains a realistic alternative.
Mathematical modeling can be combined well with dual centrifugation, but they warned us that such modeling requires high computing power.

Implementation
For our next experiments, we plan to use the cationic lipid DOTAP, but we will probably use this lipid in combination with a neutral lipid, DOPE, to ensure encapsulation as efficiently as possible while trying not to damage mammalian cells. We are planning experiments to test this approach. We will also integrate our plasmids during rehydration and try to encapsulate our plasmids in liposomes for the first time. We will also try extrusion, as it seems to be the most effective method for downsizing liposomes after Bender, Ruppl, and Römer have explained it to us. We are getting support from the Signalhaus in Freiburg, where our laboratory is based.

Outlook
We are considering the possibility of using Gb3 to modify our liposome surface. We want to read more literature for future applications. As we are not planning to manufacture hardware, we just want to find out about a suitable application. Based on the information from Bender and Ruppl that ingestion with a nebulizer is rather difficult to implement, we definitely have to continue looking at intravenous and inhalation applications.

Key Implementation

• Adjust flow rate and flow rate ratios of lipids in ethanol and DNA in aqueous buffer to significantly increase encapsulation efficiencies compared to the pipetting method, demonstrating the potential for improvement with microfluidic mixing.

Purpose
We got in contact with Dr. Jacob Hess, Head of Microfluidic Platforms at the Hahn-Schickard Institute in Freiburg. As we were looking for a method to upscale the LNP production, microfluidic mixing showed promise. With the help of Dr. Hess' resources, we hoped to find a new way to efficiently produce LNPs

Contribution
On our first visit, we were introduced to the Nemesys system which can be used to adjust flow rates and volumes applied to a mixing platform from multiple syringes. In our case we were planning to use two syringes, for each the lipid solution and the plasmid solution. Following the recommendation of Dr. Hess, we began searching for a mixing platform at Microfluidic ChipShop. After some research of our own we chose the Herringbone Mixer Fluidic 1460, which already had an application note for the production of LNPs. To start with we used a similar setup as in our pipetting experiments, adapting the volumes, concentrations and flow rate ratios from two different sources: First the application note for the chip and second a paper describing both pipetting and microfluidic mixing setups. In the lab we then adjusted the syringes and successfully formed LNPs using microfluidic mixing. In that process Dr. Jacob Hess and Trong Nguyen helped to gain access to the laboratory, initialize the system, showed us how to adjust the parameters and assemble the microfluidic tubing.

Implementation
The resulting particles were measured in the DLS and DNA encapsulation was assessed with the PicoGreen assay. DLS results were similar to our pipetting results, however the encapsulation appeared less efficient so we returned to the lab with adapted protocols. This time we tried different flow rates and flow rate ratios. We also used the same total amount of pUC19 plasmid and lipids (DOTAP, DOPE, POPC) as calculated for our finalized ratio. With the adjustments encapsulation efficiencies appeared much higher than with the pipetting method, showing that with the right changes to the parameters microfluidic mixing poses great room for improvement.

Outlook
With the kind support of Dr. Hess, Trong Nguyen and Microfluidic ChipShop we were able to implement an LNP formation method suitable for upscaling, better replicability, advanced size control and higher encapsulation efficiencies. These are important aspects when considering the actual application of our concept for industry and drug production. All this was achieved in a suitable setting for research purposes, adjusted for the scale of our project.

Key Implementations

• Produce lipid-based nanocarriers through microfluidic mixing (an effective method also for scaling up CAPTURE production beyond lab bench).
• Continue to utilize PicoGreen assays, as it is a standard method for encapsulation assays.

Purpose
Cytiva is a company providing various materials and devices for the formation and processing of lipid-based nanocarriers. Reaching out to them seemed helpful as they have experts and experience especially in the field of microfluidic mixing. Thus our aim was to learn about alternative lipid-based nanocarrier formation methods and microfluidic mixing as a method suitable to upscaling the lipid nanoparticle production for drug production.

Contribution
Dr. Ramasamy provided us with an introduction of LNP formation in microfluidic mixing and the terminology of lipid-based nanocarriers like LNPs, lipoplexes, liposomes and their use in drug delivery. We discussed our experimental setup with the PVA swelling and learned that going forward from a proof of concept ionizable lipids should be used instead of cationic lipids as they are less cytotoxic. Controls must be performed by simply pipetting the lipids on the target cells to compare effects of the drug and effects of the lipids alone. We learned that we should not neglect LNPs over liposomes as they are also very suitable for transformation of bacteria. A simple experiment to form LNPs would be to pipette the lipids and the pDNA together directly. For encapsulation assays he further encouraged us using the Ribo/PicoGreen assays. When mixing pDNA and Lipids he reminded us to consider the surface charge, and size of the pDNA which should work best below 10 kb. In the end he forwarded us the contact of a Cytiva field application scientist in Germany, Dr. Janin Germer.

Implementation
We learned to differentiate the various lipid/DNA complexes resulting from our experiments. Also we learned flaws of our setup at the time which we have to consider in the case of moving on from a proof of concept. With the knowledge about standardized lipid compositions and formation methods we discussed our setup against them and considered alternatives for example to the PicoGreen assay.

Outlook
We then reached out to Dr. Janin Germer to discuss practical aspects of our setup in more detail.

Key Implementations

• Adapted RNA encapsulation protocols and lipid ratios for our lipid formulation and pDNA encapsulation.
• Learn about common methods and novel approaches in LNP characterization.

Purpose
After receiving the information that we will have the possibility to test microfluidic mixing with the working group of Dr. Jacob Hess at the Hahn-Schickard-Institute we also got in contact with Bernhard Kirchmair, a master’s student of physics who was working on “RNA Quantification in Lipid Nanoparticles Using Fluorescence Correlation Spectroscopy” [B. Kirchmair, Master Thesis, Ludwigs-Maximilians-Universität, Faculty of Physics, Munich, March 2024]. This gave us the opportunity to learn more about practical aspects, useful protocols and measurement techniques in the field of microfluidic mixing.

Contribution
For the LNP formation usually standardized lipid composition of ionizable lipids, PEGylated lipids and Cholesterol as a helper lipid were used, making it necessary to adapt protocols for our lipid composition. To do this it is important to consider the N/P ratio, which consists of the amount of nitrogen from the cationic/ionizable lipids and amount of phosphate depending on the size and concentration of the plasmid. For RNA it is usually set to 3:1, adapting to DNA it should be 3:1 as well, if the concentration determined as mass per volume stays the same. For choosing the chip, it is important to note that most chips can not usually be reused, depending on the device. This was helpful while planning the experiment and finding sponsors for materials. This is especially the case for devices designed specifically for the purpose of LNP formation. Using simpler, modulative microfluidic mixing platforms, one could consider washing the chip with ethanol and water. However residue droplets might interfere with the mixing process. Characterizing the LNPs afterwards can be done with various measurement techniques. For DLS interpretation it is important to note that usually the intensity distribution is shown and the conclusive results are the average diameter and the polydispersity index. Also a RiboGreen or PicoGreen assay is standard to determine the encapsulation efficiency before actually quantifying the RNA/DNA per nanoparticle, depending on the size. We learned that the measurement method used in the Master Thesis, Fluorescence Correlation Spectroscopy, could be implemented with a confocal microscope as a basis, which would also have been at our disposal and can be discussed as an advanced characterization method in further experiments.

Implementation
For our microfluidic mixing experiments we paid attention to calculate the respective lipid/dna ratios and concentrations, depending on helper lipids and cationic lipids in our formulations. We considered the setup with concentrations and flow rates as stated in the protocol from the masters thesis. Learning the size of the particles might correlate with the amount of encapsulated plasmid we also continued with further characterization experiments focussed especially on this aspect.

Outlook
After this meeting we were set to start with microfluidic mixing, as materials and basic concepts were now clear and available for us. Next we had to build and test the microfluidic-mixing setup in the Hahn-Schickard lab.

Key Implementation

• We were able to include microfluidic mixing in our lab workflow to upscale LNP production.
• We drew inspiration from established protocols of Dr. Germer producing mRNA-encapsulated LNPs, which we adapted to create LNPs loaded with pDNA using the pipetting method.
• Verify an effective approach to demonstrate the encapsulated pLNPs with gel shift technique.

Purpose
After realizing there is still a lot to learn about lipid based DNA carriers and that we could use help to orient ourselves in this research field, contacting Dr. Janin Germer as a field application scientist provided the opportunity to benefit from her experience of handling with LNPs and gain access to protocols and practical tips.

Contribution
Initially, Dr. Janin Germer underlined the information we already received from Dr. Mohan Ramasamy. We should consider LNPs rather than liposomes and ionizable lipids rather than cationic lipids. Additionally, we learned that in lipoplexes the DNA is not necessarily only complexed on the outside of the liposomes. We then asked if it was possible to prepare LNPs with a simplified microfluidic mixing protocol, since we did not have the infrastructure at the time. For this we learned in more detail the basic principles in microfluidic mixing: dissolving lipids in ethanol, whereas RNA and thus DNA too are dissolved in an aqueous buffer. Both solutions are then usually mixed in a 3:1 volume:volume ratio by various methods including simple pipetting but also vortexing or in microfluidic channels of various structures. All those forms of mixing include an important step, the dialysis to get rid of residue ethanol in the sample. Here an alternative could be centrifugation to wash the LNPs. Aside from LNPs and microfluidic mixing, we discussed options to prove encapsulation in our PVA swelling formed liposomes and purify residue DNA in the surrounding solution. For example with a gel shift to separate liposomes and unencapsulated DNA. However, we realized the charge applied to the gel might compromise the liposomes, other than with LNPs where a gel shift can work well. Also the liposomes might get drawn into the gel. Another option might be a simple Nanodrop measurement, however it would be difficult to differentiate DNA outside and inside of the liposomes. When considering microfluidic mixing with the right settings the encapsulation efficiency is very high, questioning the relevance of a purification of residue DNA before application since residue DNA will usually not transform bacteria if they are not competent.

Implementation
We received a paper with various mRNA loaded LNP formation protocols which we used as inspiration to form pLNPs with the pipetting method. The meeting further encouraged us to include microfluidic mixing in our workflow. It also helped us narrow down our different approaches, pausing gel shift assays until we form LNPs and further considering fluorescence quantification assays with the plate reader or labeling the DNA directly.

Outlook
We initially put this branch of the project on hold as we were lacking infrastructure and material to proceed with. However, when we made contact with the AG Hess at the Hahn-Schickard-Institute Freiburg, the information we received became important background knowledge to plan our microfluidic mixing experiments.

Key Takeaway

• Chose not to pursue Clearcoli^TM ΔnlpI strain due to both licensing restrictions and lower OMV production rates. Further research was required in order to choose a more suitable OMV producing bacteria strain to use.

Introduction
Prof. Dr. David Putnam conducts research within the fields of vaccine development, controlled drug delivery & biomimetic polymers.
His group utilized OMVs extracted from the E. coli BL21(DE3) Clearcoli strain as biosafe vaccine platforms [Weyant et al. 2023].
The Clearcoli^TM strain has been engineered to contain modified lipopolysaccharides that induce a significantly lower immune response compared to the wild type strain.

Purpose
Our first aim was to find a suitable strain for our OMV production. Preferably, with the deletions necessary for hypervesiculation alongside modifications for reduced immunogenicity.
To us, the Clearcoli^TM ΔnlpI strain used by his group fulfilled all our criteria for an OMV producing strain. However, Clearcoli^TM is owned by Research Corporation Technologies (RCT) and we were concerned about licensing issues that may prevent future teams from following in our footsteps. Therefore, one of our questions was about the legality of simply using the Clearcoli^TM ΔnlpI strain.

Contribution
According to both Prof. Putnam and our primary investigator Dr. Pavel Salavei, the modifications have indeed altered growth characteristics, with slower growth that could also reduce the overall rate of OMV production.
We were also informed that the Clearcoli^TM strain had a strict licensing policy attached, limiting the potential exchange of this strain and its results with other iGEM teams. This was incompatible with our vision of “Open Source Synbio”.

Outlook
Due to the following reasons, we decided to not go forward with the Clearcoli^TM ΔnlpI strain:

• A strict license agreement would be a significant roadblock in the way of cross-team collaboration and or commercialization.
• Generally weaker growth characteristics resulting in longer doubling times & reduced OMV production.

We therefore continued our quest for the 'OMV producing strain'.

Key Implementation

• Chose E. coli omp8 strain for OMV production and experiments due to promising OMV yield.

Purpose
Prof. Dr. Daniel J. Müller chairs the Biophysics department at ETH Zurich. His research group specializes within the field of Biophysics, focussing on the development of biophysical tools to quantify and evaluate cellular interactions and membrane receptors.
His team utilized OMVs derived from the E. coli BL21(DE3) omp8 strain for outer membrane protein studies [Thoma et al. 2018].
Recombinant outer membrane proteins could be readily expressed on OMV surfaces, allowing for further studies with the expressed outer membrane proteins.
While the BL21(DE3) omp8 strain used had the necessary ΔompA, ΔompC gene knockouts required to fulfill the hypervesiculation criterion, we were skeptical about its general growth characteristics. We therefore contacted Prof. Müller to assess potential increases in doubling time alongside the feasibility of mass-culturing this strain as part of a plan to mass-produce OMVs.
Our second question was regarding licensing, namely if our team would be allowed to share a further modified strain with other iGEM teams without having to go through restrictive legal hoops.

Contribution
According to Prof. Müller, the strain did not show a significant deviation from normal growth characteristics observed with wild type E. coli BL21(DE3) strains. However this may be derived from the deletion method employed; silencing mutations as opposed to complete genomic deletions, which did in fact cause significant reduction in cell growth characteristics [Meusken, et al. 2017]
With regards to licensing, Prof. Müller explained that the strain was able to be used by further iGEM teams so long as we cited the strain’s source [Prilipov, et al. 1998]

Implementation
We decided to go forward with the omp8 strain for OMV production due to the following reasons:

• Overexpressed recombinant outer membrane proteins constituted a greater portion of all outer membrane proteins in omp8 compared to the wild type strain.
• Growth characteristics were not significantly altered due to the four gene knockouts.
• E. coli BL21(DE3) omp8 does not require licenses, allowing us to collaborate with present and future iGEM teams.

We were still skeptical of its immunogenicity, as a result of which we did further research on strategies to minimize an immune response within the patient.

Key Takeaway

• Choose to not use biotin binding domain due to high nonspecific binding which would not align with our project’s biosafety values.

Purpose
Prof. Mathew DeLisa at Cornell University’s College of Engineering, conducts research in the fields of protein folding & assembly, protein-translocation & post-translational modifications.
His team specializes in the underlying mechanisms involved in protein biogenesis. They utilize the Lpp-OmpA outer membrane protein bound to a biotin binding domain; Avidin for the exhibition of biotinylated antigens on the OMV surface [10]. Simply put: Biotin fused antigens could successfully bind to the OMV surface via Avidin.
Our second task was to find a suitable “universal adapter” to bind any suitable targeting ligand onto the OMV surface, ensuring specific targeting to P. aeruginosa. We believe that Avidin along with Biotinylated antigens would fit this role well. Therefore, we contacted Prof. DeLisa to enquire about the mechanisms in place with regards to specificity between the biotinylated antigen & the binding domain exhibited on the OMV surface.
We also want to narrow down our outer membrane protein candidates onto which we would fuse our “universal adapter” and with it, our targeting ligands. We felt that Prof. DeLisa with his expertise in protein translocation could help us significantly with this.

Contribution
According to Prof. DeLisa, non specific binding was not observed when enhanced monoAvidin was used in tandem with the Lpp-OmpA membrane anchor. However, despite its good functionality and display the overexpression of this membrane anchor significantly affected cell viability.

Outlook
After a meeting with one of our primary investigators, Dr. Pavel Salavei, we decided to not use biotin binding domains in our project. Our reasoning stems from the fact that biotin binding proteins such as Avidin tend to have high non-specific binding, which would be a detriment to our project’s biosafety [Balzer et al. 2023]. This means that we have to continue looking for alternatives for our “universal adaptor”.

Key Implementation

• Used SpyCatcher-SpyTag system as the 'universal adapter' due to its high specificity and strong binding affinity. Having decided on the “universal adapter”, we then focussed our efforts on finding a suitable outer membrane protein for its display on the OMV surface.

Purpose
Prof. Dr. Jian-Dong Huang holds the chair for Biomedical Sciences at the University of Hong Kong. His research interests include the utilization of synthetic biology for the development of vaccines against infectious diseases & novel cancer treatments.
His team adapted the SpyCatcher-SpyTag system system for antigen display on the OMV surface [Sun et al.]. Simply put: SpyTag functionalized antigens could bind to SpyCatcher bound OMVs.
We contacted Prof. Huang to enquire about the specificity & binding strength of the SpyTag-SpyCatcher interaction alongside general questions regarding the biosafety of such vaccines. In a further step, we wanted to know if there were any methods to decrease the immune responses associated with outer membrane vesicles.

Contribution
According to Prof. Huang, the covalent bond formed between the SpyTag peptide and the SpyCatcher protein is both extremely strong and specific, this fact would effectively invalidate non-specific adsorption while allowing for an extremely biosafe surface modification strategy.
In addition, Prof. Huang suggested a plethora of deletions that one could perform to attenuate endotoxicity and immunogenicity associated with OMVs: ΔmsbB, ΔpagP, ΔhtrA, ΔclpPΔphoP/phoQ, ΔaroA, ΔguaBA, ΔclpB, ΔlpxM, ΔdsbA, Δwzy.

Implementation
In light of its high specificity & strong binding affinity, we decided to use the SpyCatcher-SpyTag system as the “universal adapter” through which we would be able to attach our targeting ligands.
After this meeting, we pivoted our efforts towards finding a suitable membrane protein for SpyTag expression.

Outlook
However, due to time constraints we were not able to integrate any of the deletions into our project. We do however plan on performing and testing out these deletions as part of our project outlook. Read more about this on our Description-Outlook Page.

Key Takeaway

• While the insights provided by Dr. Rabe were compelling, our team ultimately decided to pursue a different membrane protein.

Purpose
Dr. Kersten Rabe is a group leader at the Niemeyer Lab in the Institute for biological Interfaces 1 (IBG-1) at Karlsruhe Institute of Technology. His interests include biocatalysts and protein engineering.
His team expressed SpyCatcher on cell surfaces by fusing it to four different outer membrane proteins. These were then evaluated on the basis of their functionality, expression level & effect on overall cell viability. The paper concluded that Lpp-OmpA showed the highest levels of expression, however with a significant reduction to cell viability.
Our questions to Dr. Rabe were centered on the outer membrane protein used on the OMVs; whether Lpp-OmpA was a suitable candidate for a genetic fusion to SpyCatcher despite its adverse effects on cell viability or if alternative outer membrane proteins had to be taken into consideration.
We also sought his opinion on which of the two partners, SpyTag or SpyCatcher, should be expressed on the outer surface.

Contribution
Dr. Rabe suggested using the PsgA and AIDA-I membrane protein based constructs instead of the Lpp-OmpA construct due to the greater membrane permeability membrane associated with the latter. However the best solution according to him would’ve been to perform a genomic alteration, so that a native E. coli protein fused to one of the two Spy partners would be overexpressed.
With regards to the specific partner expressed on the membrane protein, Dr. Rabe placed more importance upon the specific partner fused with the phage tail, as the translocation of the membrane anchor isn’t affected much with a change of the Spy partner which when paired with a larger protein such as SpyCatcher could possibly cease to function. It is for this reason he suggested we not choose a SpyCatcher fused phage tail.
Regarding the specific partner expressed on the membrane anchor, Dr. Rabe placed more importance upon the specific partner fused with the phage tail, as membrane anchor translocation wouldn’t be significantly altered by a change in the fused Spy partner.

Outlook
We decided to not go forward with PsgA and AIDA-I, in favor of an Lpp-OmpA construct for which we contacted the DiVentura Lab as they had previously obtained a sample of the same.

Key Implementation

• Chose eCPX as the outer membrane protein candidate for fusion with the SpyTag peptide, as a part of the modular approach towards targeting ligand functionalization, due to its reduced effects on cell growth and viability alongside high peptide display rates.

Introduction
Büşra Merve Kırpat Konak is a PhD student at the Di Ventura research group in the University of Freiburg’s Faculty of Biology. After having contacted Dr. Rabe of access to the Lpp-OmpA-SpyTag fusion coding plasmid, he directed us to the Di Ventura research group at our own university.

Purpose
Ms. Kırpat Konak has previously researched and compared different outer membrane proteins as part of her project to express genetically fused proteins on the outer surface of bacteria. This tied in very neatly with our project. We wanted to genetically fuse SpyTag to an outer membrane protein to act as our “universal adapter” onto which our SpyCatcher functionalized targeting ligands could fuse.
Our goal was to gain more insight into outer membrane proteins that could be overexpressed without inhibiting cell growth by virtue of overloading protein translocation machinery, which could then be used to exhibit SpyTag on the OMV surface.

Contribution
After meeting with Ms. Kırpat Konak, we were given 3 different plasmids that encoded a fusion protein consisting of eCPX outer membrane protein and the SpyTag peptide. According to Ms. Kırpat Konak, the eCPX outer membrane protein would be better suited for our needs without causing reductions in cell viability.

Implementation
We utilized the pTrc99a backbone-based eCPX-SpyTag encoding plasmid (given to us by Ms. Kırpat Konak) for SpyTag display on the OMV surface.

Key Implementations

• For the cell culture-based experiments we chose lung cells instead of HEK cells, as CAPTUREs mode of action is in the lung.
• For the future development of CAPTURE, we have considered aerosol spraying through a nebulizer for a possible treatment application in the lung.

Purpose
Dr. Peter Walentek and his workgroup focus on the molecular mechanisms of cilia and mucociliary epithelium. They are well-versed in lung organoids and the implementation and execution of experiments with and on lung cells. With his help, we want to find out how we can best carry out experiments without animal testing and how we can best imitate the mucosa to prove CAPTURE. We also want to ask for his general expertise on our project. His expertise in future implementations of CAPTURE in hospitals is also valuable due to his proximity to Freiburg University Hospital.

Contribution
At the beginning, we asked him how we could best cultivate and process mammalian cells. He is convinced that healthy cultured cells survive longer in PBS and that there should be no lysis. He also recommended that we clean the cells with FBS if we no longer wanted to continue working with PBS. He also suggested that we use lung cells instead of HEK cells to get a better proof of concept. Calu-3 cells are easy-to-cultivate lung cells that almost every laboratory working with cell cultures can use.
Also interesting for our experiments are Air Liquid Interface Cultures, which, as Walentek explains, are cells from donated lungs. We should read up on this further. He suggests selected literature for this. We should also use a suitable reporter gene that individually marks all CAPTURE, encapsulation, and fusion processes. This allows us to intervene more effectively and find reasons and solutions to problems more quickly. If our Sushi S1 might not work well enough, he suggests aquaporins. We can use them to produce similar membrane pores.
Walentek believes that the introduction of liposomes into the lungs through a nebulizer will definitely work. However, he cautions that it is very difficult to bring to the market. The path to a finished, prescription drug is an obstacle for many researchers, even those with experience. According to him, animal experiments cannot be avoided. Experiments for lung drugs are often carried out on ferrets, as they are the closest to human lungs. He also suggests that we should illustrate the nebulizer on our graphical abstract so that viewers have an idea of how CAPTURE can be introduced. For a good proof of concept, he offers us the opportunity to test mucus-like texture in his laboratory, which is produced from frog embryos. We can drip our cargo with the carrier onto the mucus and see how it behaves. In the building where the meeting took place, the IMITATE (Institute for Disease Modelling and Targeted Medicine) Freiburg, we also found a guide for start-ups in the southwest of Germany. It is called Impact Start-ups, a brief guide through a startup ecosystem by the urban management marketing of Freiburg and Startinsland, a startup alliance in the Freiburg area.

Implementation
We will now investigate the cause of our prematurely dying HEK cells in PBS in the laboratory. We have also received new and more suitable plates. We will use them to repeat the previous expression series and determine whether the problem was related to our vessels. We will definitely take his experience in applying for animal testing and implementing medical products on the market into account in CAPTURE. In particular, the idea of administering CAPTURE with a nebulizer is a good one, but difficult to implement. We are aware that this step would not take place within the framework of iGEM, but we already want to explore how we can best prepare CAPTURE for the market.

Outlook
We are now also planning experiments with lung cells, as we believe that experiments with lung cells will have a more lasting impact on our proof of concept. We are certainly interested in accepting his offer to apply CAPTURE to the mucosa of frog embryos in his laboratory, provided there is sufficient time available. Additionally, in preparation for clinical implementation, we would like to explore potential funding opportunities and collaborative partnerships.The meeting with Walentek turned out to be a double stroke of luck, enabling us to improve both our laboratory work and our commercialization efforts for the future.

Key Takeaway

• Explore the potential of Electron Microscopy to visualize the effects of the AMP Sushi S1 on the cell membrane of P. fluorescens, given its high magnification capabilities (eventually, due to the significant costs and time required for this technique, we decided not to use it for further screening of our transport vesicles).

Introduction
Dr. Martin Helmstädter is the head of the Electron Microscopy Core Facility IMITATE in Freiburg. As part of the university, the Core Facility IMITATE offers a broad spectrum of scanning and transmission electron microscopy methods for research groups around Freiburg.

Purpose
Since we have now produced the first outer membrane vesicles and lipid based nanocarriers and cultivated first colonies of P. fluorescens, we are curious how they appear in light. Due to their size of about 200 nm and smaller, it is not possible for us to look at the particles in detail with conventional microscopes in our laboratory. We have therefore contacted Dr. Martin Helmstädter to get a rough overview of the possibilities and opportunities of electron microscopy.

Contribution
We learned that the best preparation method for screening the bacteria under the electron microscope is to rehydrate them on a thin glass slide. To screen OMVs or/and Lipid-based Nanocarriers, Dr. Helmstädter advised to fix the transport vesicles with glutaraldehyde. As glutaraldehyde is toxic, he provided us to do the fixation of CAPTURE transport vesicles for us.

Implementation
Since the electron microscopy is expensive and time consuming, we decided to not screen our transport vesicles or/and P. fluorescens via electron microscopy.

Outlook
Electron miscroscopy offers opportunities to show effects of the Antimicrobial Peptide (AMP) Sushi S1 in the cell membrane of the bacterium P. fluorescens due to its high magnification. As we do not yet have a Sushi S1 encoded on a plasmid, we can only consider this method for the future.

Key Takeaway

• Gained insights on contamination prevention in hospitals and discussed the future outlook and clinical application of CAPTURE.

Purpose
Our second modeling part aims to understand bacterial spread in hospitals and lungs. Dr. Tjibbe Donker’s working group at the University Medical Centre Freiburg specializes in quantitative and predictive disease epidemiology. His research focuses on antibiotic resistance and bacterial distribution on surfaces in hospitals. Our aim was to learn more about bacterial and antibiotic resistance multiplication rates and to explore ecological aspects of bacterial reproduction. Accordingly, we want to know how quickly the bacterium and its antibiotic resistance can multiply. We also want to look at the ecological aspects of bacterial reproduction in detail in order to find ideas to make our project even more efficient.

Contribution
Dr. Donker shared a valuable case study on P. aeruginosa contamination in a hospital for pulmonary patients. Due to the minimal growth requirements of P. aeruginosa, the pathogen can thrive in diverse environments.
But what does that mean? Dr. Donker highlighted several key points: He explained that many pathogens naturally accumulate in sanitary facilities, such as washbasins and their pipes. Patients need to wash themselves or be washed. Water pipes and sanitary facilities are interconnected. P. aeruginosa is an aquatic pathogen that can also grow anaerobically and tends to accumulate in biofilms within the pipes. He also explained that during the normal use of water taps, water is deposited as tiny particles on the drain. Nanoparticles, pathogens, and the water itself can spread throughout the room in a radius of several meters, leading to contamination via the water supply. Patients with similar diseases often live in the same ward in the hospital. P. aeruginosa spreads through water channels in the hospital, so it is common for patients in the same ward to be infected with the same type of P. aeruginosa. He also recommended consulting Dr. Irina Nazarenko, an expert in extracellular vesicles, for further insights.

Implementation
Our discussion with Dr. Donker reinforced CAPTURE’s potential as a targeted therapy against P. aeruginosa in lungs, particularly in hospital settings where similar strains often infect multiple patients. Donker thus confirmed to us that CAPTURE can fight infections at a specific point in time for a uniform patient group. In response to the insights gained, we will:

• Conduct a thorough cleaning of our laboratory.
• Reorganize our sanitary facilities by areas of responsibility.
• Implement preventative measures to avoid contamination.

We will carry out a thorough cleaning of our lab and divide our sanitary facilities according to areas of responsibility. In this way, we want to take preventative measures to avoid contamination.

Outlook
We will contact Dr. Irina Nazarenko to discuss Outer Membrane Vesicles (OMVs).

Key Takeaway

• For future experiments, Dr. Wohlwend suggested applying enzymes Dpnl and Proteinase K to digest genomic DNA, RNA, and native proteins in OMVs. This could increase the significance of prospective experimental results.
• While our LNPs and OMVs were too large for the mass photometry device to measure their mass of individual particles, it still allowed us to obtain images of our LNPs and OMVs.

Purpose
Dr. Daniel Wohlwend, head of a Biochemistry Group at the University of Freiburg, introduced us to mass photometry, a technique for measuring the mass of proteins and vesicles. We aimed to explore whether this method could detect and evaluate the mass of our lipid nanoparticles (LNPs) and outer membrane vesicles (OMVs).

Contribution
Dr. Wohlwend explained how mass photometry uses laser light to visualize proteins and other particles by detecting light waves reflected by the particles and approximating their molecular weight through comparison with calibrated standards. We were given the opportunity to test the method on our empty LNPs and OMVs. Unfortunately, the size of the vesicles was too large for the device to handle, and background noise from other particles interfered with detection. Additionally, the OMVs adhered to the glass surface, further complicating the measurements.
Despite these limitations, the experiment produced clear images of our vesicles. In the discussion that followed, Dr. Wohlwend suggested using DpnI and Proteinase K to digest genomic DNA, RNA, and native proteins within the OMVs, leaving behind empty OMVs. He also recommended using Lipase instead of TritonX-100 to destroy the OMVs and LNPs while preserving the plasmid.
We also discussed how to deactivate β-lactamase within OMVs by heating them to 95°C for 10 minutes to avoid interference in future ampicillin resistance experiments.

Implementation
While mass photometry may not be suitable for measuring larger vesicles, the insights gained will guide us in improving sample preparation and fusion experiments.

Outlook
In future experiments, applying DpnI and Proteinase K could help generate empty OMVs for more precise testing. Lipase treatment may also be explored as a method to selectively degrade OMVs and LNPs while preserving intact plasmids. Additionally, thermal deactivation of β-lactamase at 95°C could be considered to avoid interference in ampicillin resistance assays.

First Meeting

Purpose
We consulted Bioethicist PD Dr. Joachim Boldt from the Institute for Ethics and History of Medicine in Freiburg to discuss ethics and safety of our project. He is an expert in ethical questions regarding synthetic Biology and medicine, and deputy chair of the Ethics Committee of the University of Freiburg. At our first meeting in February we had not decided on the project CAPTURE yet. But no matter the direction of our project, we knew that it was important to us to be aware of any ethical issues that could potentially arise and take precautions before starting the journey of CAPTURE.

Contribution
Dr. Boldt has been engaged with iGEM since 2004 and has been working in the field of synthetic biology for a long time. Therefore, he knew where we stood and gave us advice on how to proceed.
Firstly, he informed us about the history of synthetic biology and outlined his research objectives. His main focus is examining how linguistic, philosophical and ethical questions evolve and become relevant in specific cases of synthetic biology.
Based on experiences of the last iGEM teams, Dr. Boldt shared valuable insights on successful approaches and areas for improvement in ethical considerations.
An important key concept throughout the meeting was Upstream Bioethics. This refers to the preparation of ethical challenges in advance coupled with an awareness of potential difficulties that may arise. This means, it is important to integrate ethical considerations while planning the project, rather than addressing them as an afterthought once the project is complete. This approach ensures that the design meets the highest ethical standards.
In the process of finding a project, Dr. Boldt advised us to keep local and global problems in mind and check whether a combination would be possible.
Since we did not have a specific project defined at the time of this meeting, we knew that we wanted to have a follow-up meeting with Dr. Boldt once our project has developed further. This will allow us to discuss potential ethical challenges specific to our project direction.
Finally, he recommended us, the human practices subgroup of our team, to maintain clear communication within the entire team and engage with stakeholders from diverse perspectives, including researches, public health organizations and legal experts. This approach will ensure a comprehensive understanding of the ethical implications of our work.

Implementation
In order to consider possible ethical challenges and to prepare for our next meeting, Dr. Boldt suggested we get back to him when our project idea has shaped further.
Our next step was to reflect on which specific issue we are aiming to address with our project and tp contact Dr. Boldt as soon as possible.

Second Meeting

Purpose
As our project evolved, we scheduled a second meeting with Dr. Boldt in April to identify potential ethical challenges CAPTURE might face, continuing our journey in Upstream Bioethics.

Contribution
We began the meeting by revisiting our team’s reflections from between the first and second meeting. Dr. Boldt endorsed our approach to combat P. aeruginosa recognizing the urgent need for antibiotic alternatives. He provided further valuable guidance on how to improve the safety of CAPTURE.
Firstly, he advised us to develop a target mechanism that specifically binds to P. aeruginosa and to explore additional safety strategies, such as promoter selection. This advice was complimentary to our initial ideas, which gave us confidence in developing a target mechanism to improve CAPTURE´s biosafety. We decided to implement a P. aeruginosa-specific promoter combined with targeting ligands, PhageTail and Gb3, as part of our strategy.
Our discussion then shifted to the optimal application of Upstream Bioethics, particularly in the context of future drug testing phases. Dr. Boldt recognized our desire to minimize animal testing in potential preclinical studies and suggested exploring lung organoids as an alternative. These three-dimensional cell culture models mimic the structure and function of human lungs, potentially reducing the need for animal trials. To gain further knowledge in this field, we planned to contact Dr. Walentek (IMITATE - Institute for Disease Modeling and Targeted Medicine Freiburg).
The final topic we addressed was the patenting of our treatment. We explored the possibility of CAPTURE as an open-source therapy, which implies reducing the cost of medications, increasing transparency between research and the pharmaceutical industry and improving access to essential medications, particularly in underserved regions. However, we have also identified a potential disadvantage to an open-source model, as there is a possibility that fewer pharmaceutical companies may choose to invest in the production and development of the treatment. To ensure the best possible implementation of CAPTURE in clinical settings, we decided to consult Prof. Grundmann. This step would help us to manage the complexities of bringing our innovative treatment from the lab to real-world medical applications.

Implementation
Throughout this follow-up meeting with Dr. Boldt, we not only addressed crucial ethical considerations but also formulated concrete steps to enhance the safety, efficacy, and ethical standing of our CAPTURE project. This meeting exemplified the ongoing nature of Upstream Bioethics, demonstrating how ethical considerations continue to shape and refine our scientific approach throughout project development.

Key Implementation

• Implement mammalian cell culture experiments with A549 cells to test for cytotoxicity and future clinical application.

Purpose
In our ongoing efforts to explore alternatives to animal testing, we had the opportunity to engage with Prof. Dr. Jens Kurreck, a leading researcher known for his work in developing organ models that aim to reduce reliance on animal experiments. Our contact with him was facilitated through the organization Doctors Against Animal Experiments. Prof. Kurreck's research focuses not only on providing better alternatives but also on advancing the search for innovative solutions. Given his expertise, we sought his guidance on how we might avoid animal testing in clinical trials and gain insights into viable animal-free research methods.

Contribution
Prof. Kurreck emphasized that using animals, particularly mice, in experiments is fundamentally flawed due to the significant differences in pathogenesis between animals and humans. He highlighted that experimental results obtained from mice are often not translatable to human biology, making such experiments scientifically unsound. Despite decades of animal-based research, he noted that mortality rates have remained constant over the past 40 to 50 years, with 90% of all drugs failing during clinical trials. This alarming statistic underscores the urgent need for models that are more appropriate and comparable to human physiology.
Kurreck's research group has successfully developed three different lung models designed to outperform traditional animal models in predicting human responses. They are also in the process of developing a model for cystic fibrosis (CF). However, Kurreck advised against using our CAPTURE platform on CF models due to their high genetic variability, which would make the results too nonspecific.
He strongly recommended the use of human-derived cell lines, such as A549 or Calu-3 cells, which can be cultured in the laboratory and provide more reliable and human-relevant data. Furthermore, Kurreck suggested that we reach out to Prof. Dr. Donald Ingber from Harvard University, who is pioneering the use of organ-on-a-chip technologies for modeling human organs and disease processes.

Implementation
Our meeting with Prof. Kurreck reaffirmed our commitment to avoiding animal testing. We were particularly struck by the revelation that many animal experiments fail to produce reliable or translatable data regarding the efficacy of treatments in humans. For our future experiments, we will shift our focus toward the use of human mammalian cells, particularly A549 cells, which have been shown to provide relevant and reproducible results in preclinical research.

Outlook
Given Prof. Kurreck's research and his focus on replacing animal testing with more accurate models, we are now considering the following steps:

• Investigating established alternatives to animal testing in clinical trials, particularly focusing on human-relevant in vitro models.
• Further exploring the challenges, benefits, and limitations of both animal experiments and alternative methods to make more informed decisions moving forward.

By pursuing these strategies, we hope to contribute to the growing field of animal-free testing methods that are not only ethically responsible but also more scientifically sound.

Key Takeaway

• Incorporate HSTII secretion signal into the AMP plasmid due to functionality of Sushi S1 in extracellular space.
• Acknowledge the importance of the Phage Tail P2 targeting system on our OMVs.

First Meeting

Purpose
Prof. Dr. Burkhard Tümmler, Coordinator of the Disease Area Cystic Fibrosis at the German Centre for Lung Research. We sought his expertise in lung research, particularly regarding the effects of P. aeruginosa infections in the lung. Given his current role and specialization, Prof. Tümmler provides valuable insights for our project. The purpose of our meeting was to present our project to Prof. Tümmler and identify potential weaknesses. We unveiled our graphic abstract for the first time to assess its effectiveness in presentations. Additionally, our aim was to understand how CAPTURE affects the body and lungs, focusing on the specificity and safety of our plasmid carriers.

Contribution
Right at the beginning of our conversation, Prof. Dr. Tümmler recommended exploring Bob Hancock's work, highlighting its foundational role in global peptide research. He emphasized that while AMPs hold promise, their short half-life poses a challenge for effective delivery to the lungs. Additionally, AMPs can be hazardous to humans due to their ability to fuse with mammalian cells. Prof. Tümmler expressed concerns that our approach of using lectins to enhance liposome specificity might not be sufficient. However, he acknowledged that our strategy of specifying our OMVs with phage tail proteins to target P. aeruginosa is promising, albeit challenging due to the vast diversity of phages and bacterial strains. The likelihood of identifying the correct phage tail protein is low but achievable with extensive research. Prof. Tümmler conducted an in-depth scrutiny of CAPTURE, questioning our understanding of the chemical composition and its effects on both the body and the pathogen. He encouraged us to broaden our research by reviewing publications from various authors to improve CAPTURE.

Implementation
For the next steps, we intend to read more publications to better understand the individual components of CAPTURE and to describe them in more detail. Our goal is to expand our knowledge and explore ways to make our system more specific and efficient. As Prof. Tümmler pointed out, we currently lack sufficient understanding to effectively improve the system. Additionally, we suspect that during the meeting and through our graphical abstract, it may not have been clear to Prof. Tümmler that our aim is to deliver the AMP encoded on a plasmid. To address this, we intend to place greater emphasis on the plasmid in our graphical abstract. To facilitate this process, we will dedicate the next two weeks to exchanging ideas with Prof. Tümmler.

Second Meeting

Purpose
The second meeting with Prof. Tümmler was held to address specific questions regarding our plasmid, targeting system, and the biofilm of P. aeruginosa.

Contribution
Prof. Tümmler highlighted several factors contributing to the instability of AMPs, including the presence of ions, salt, microbial and host proteases, bacterial polysaccharides, etc. He noted that a plasmid is inherently more stable than synthesized AMPs, eliminating production costs since the bacteria synthesize the peptide themselves. He reviewed our arguments for using a plasmid instead of a synthesized AMP. In terms of choosing a specific AMP, he recommended selecting a peptide that inhibits transcription or translation. This is because the Sushi S1 AMP creates pores in the outer membrane, so once synthesized in the bacteria, it would require a secretion signal. He theorized that using an AMP with synthesis inhibition would prevent the bacteria from developing resistance due to the fast and efficient mechanism. When we asked about the possibility of cross-resistance to our AMP, he indicated that he could not provide a definitive answer. He addressed the anticipated challenge of P. aeruginosa biofilm presence, assuring us that the biomass in lungs infected with this pathogen is primarily composed of goblet cell mucus, with only a small percentage formed by Pseudomonas. Concerning our targeting mechanism for OMVs, he noted that the longer P. aeruginosa remains in the lung, the greater the chance of degrading the phage receptors and developing resistance. However, he emphasized that the diversity of phages provides a high probability of identifying one effective against Pseudomonas.

Implementation
Following the discussion with Prof. Tümmler, we decided to incorporate a secretion signal, HSTII, to transport our AMP to the outer membrane where it would target the membrane of the pathogen.

Outlook
To gain a deeper understanding of cross-resistance and its impact on P. aeruginosa developing AMP resistance, we decided to contact Prof. Dr. Rieg, an expert in antibiotic resistance and a professor at the University Hospital.

Key Takeaway

• Contact University of Freiburg’s foundation office to gain knowledge on further possibilities of patenting and founding a start-up.

Purpose
With CAPTURE gaining recognition as a novel advancement in drug research, we sought advice on patenting and market implementation. We consulted Prof. Dr. Ralf Reski, a patent expert at the University of Freiburg, known for his research on mosses and his experience in filing multiple patents.

Contribution
Prof. Reski, who attended our Faculty Day presentation, affirmed that CAPTURE is innovative and recommended securing a patent, as no similar treatments for P. aeruginosa exist. He outlined the patenting process in three phases: preparing an application, contacting the Central Office for Technology Transfer, and working with a patent attorney to secure validation at various levels (national or global). Prof. Reski offered to assist us with future inquiries or applications.

Implementation
We are considering patenting CAPTURE and may consult Prof. Reski for guidance. However, we are unsure if a patent is the best step due to uncertainties about the project’s future after the Jamboree. Additionally, we are planning to publish an abstract in BioSpektrum, but may delay it if we pursue a patent.
Key steps:

• Decide as a team whether to pursue a patent.
• Draft the patent application if necessary.
• Coordinate with BioSpektrum on publication timing.

Outlook
We are also exploring the potential of founding a start-up and plan to contact the University of Freiburg’s foundation office for more information.

Key Takeaways

• Decided not to further pursue options for patenting CAPTURE and instead put all of our attention and time into our iGEM project and the Grand Jamboree.
• gained insights into founding a start up and industry opportunities for CAPTURE´s future.

Purpose
The start-up office is one of the University of Freiburg's organizations that looks after innovative ideas on campus. There you can get advice on founding, patents and the potential of a project.
We wanted to do the same and therefore contacted the Freiburg start-up office, to talk about the potential obstacles during the establishment and resilience of our project.
At the start-up office, we had an appointment for start-up advice with Dr. Thomas Maier, who had experience with his own start-up, and Christoph Mårtensson, who had experience as an investor.

Contribution
Right at the beginning, they emphasized that AMR-related infections will be a major challenge in the future. This means that the market for new products will soon be large. Soon, because there are currently still few new treatment options and therefore little competition. The problem of AMR-related infections is not yet so well known, which is why the market and research still want to stick with the established treatment methods: antibiotics. For this reason, they say it is an important and good idea to patent CAPTURE, as it is not known how much the demand for new treatment options will increase in the coming years. As CAPTURE offers a broad spectrum of specification options that could combat not only P. aeruginosa but also other ESKAPE pathogens, it is not out of the question that CAPTURE would at least have the potential to establish itself in clinical trials. The experience of establishing a patent and founding a start-up is also a teaching experience in life, so they strongly encouraged us to turn CAPTURE into a start-up.
According to Dr. Maier and Mr. Mårtensson, it is important to act, because after the first publication in journals or as a paper, the idea is freely available to everyone and can be used by anyone without a license.
Finally, they offered to help us practice our Jamboree presentation by listening to it and giving feedback.

Implementation
We are happy to have talked about the establishment processes.

Outlook
We gave a lot of thought to whether we as a team could manage a patent in the short time available. In the end, we decided that we would prioritize the Grand Jamboree for our academic degree and not patent CAPTURE. However, the meeting was very rewarding for us, as we have already gained an insight into the possibilities of patenting products and establishing them in the industry for our future careers.

Key Takeaway

• Insights into how CAPTURE could be implemented into clinical practice.

Purpose
Prof. Dr. Hajo Grundmann has a background in clinical tropical medicine, medical microbiology, hygiene, and environmental medicine. He was the project leader of the European surveillance system for antimicrobial resistance. Through this meeting, we hope to get a general assessment of our project and gain insight into hospital work worldwide, including potential differences and their significance. With his background in microbiology, we want to know whether our targeting methods are effective and marketable. Furthermore, we seek input on considerations for integrating CAPTURE into clinical practice.

Contribution
Prof. Grundmann opened by noting that the CAPTURE idea is a new method for treating P. aeruginosa. He believes that CAPTURE can be effective because our method intervenes from the inside. Resistance develops in the periplasmic space, so our project idea seems to him to treat precisely in the patient. He also mentioned that P. aeruginosa is a very important and difficult-to-control pathogen that has many variants and can develop multiple resistances within hospitals, called high-risk clones, which are supported by its large genome. The pathogen protects itself with a biofilm, which makes it even harder to combat. Thanks to Prof. Dr. Walentek, we know that the biofilm does not play a major role, as it forms in the lungs as the tunica mucosa respiratoria. A well-known and insidious disease is cystic fibrosis (CF), which produces thick mucus in the lungs.
Based on Grundmann's many years of clinical experience, he drew attention to the problem that chronically ill patients, such as CF patients, are often victims of this pathogen. Not only is the individual perspective on pathogens important, but also the systemic one. Such patients are very costly for hospitals and healthcare systems, both in time and money. Current treatment options are lengthy and inefficient. Bacterial infections are typically treated with traditional antibiotics, which are effective but present challenges. They often lack precision in targeting specific pathogens and can disrupt both harmful and beneficial bacteria, negatively affecting the natural microbiota. This places patients in a position that may be perceived as a burden on the healthcare system, for better or worse.
With CAPTURE, we use specific targeting options for the bacterium P. aeruginosa, according to Grundmann, this is our selling point. In contrast to previous antibiotic therapy methods, CAPTURE can specifically find and kill the bacterium. Cells in the surrounding environment are not affected. In order to prove this experimentally, we suggested using the lung organoids created by Walentek. Grundmann thought this was a good idea for testing CAPTURE. Administration via nebulizer is also a good option. For intensive care patients, he could imagine an adapter that administers CAPTURE during artificial respiration in addition to ventilation. Grundmann also noted that real-time sequencing can quickly identify high-risk clones, enabling faster, personalized treatments combined with our modular targeting system. However, the high cost of sequencing machines and infrastructure limits widespread use, though prices have decreased significantly over time. Combining CAPTURE and real-time sequencing could offer a precise and fast way to treat bacterial infections.
Grundmann later drew our attention to hospitals worldwide through his experience in several hospitals in the Global South and North. He emphasized that there is far too little research on hospital conditions in the Global South, so it is not possible to conclude that hospitals there are less hygienic. According to Grundmann, this is a myth and an important point in the debate.

Implementation
We want to highlight the fact that research on infections in the Global South is often neglected. The focus is placed on infections in the Global North, and infection figures in the South are often overseen. We have also become even more confident about administering CAPTURE by inhalation, now that we also know how to administer CAPTURE to intensive care patients in a compliant manner. We are also considering ordering lung organoids from Prof. Dr. Walentek, if time permits, to test CAPTURE's effect on lung cells.

Outlook

• Research on infection studies in the Global South.
• Planning experiments with lung organoids.

Key Takeaway

• For CAPTURE's future development: verify nebulizer as a viable CAPTURE treatment delivery platform and consider CAPTURE application not only instead of, but also complementary to antibiotic-based treatments.

Purpose
Prof. Rieg is a specialist in internal medicine at Freiburg University Hospital, focusing on host-pathogen interaction and systemic therapy in humans. His expertise is particularly in sexual and vector-borne diseases affecting humans. With CAPTURE, we aim not only to achieve a proof of concept but also to progress towards clinical application. Talking to Prof. Rieg, we seek to identify potential challenges and problems with CAPTURE at an early stage and develop solution strategies.

Contribution
Prof. Rieg has significantly enriched our understanding of P. aeruginosa infections, emphasizing the risks faced by patients who acquire this infection during hospital stays. He highlighted the importance of hospitalization duration as a critical factor in the likelihood of contracting P. aeruginosa. This means that people with lung infections, such as those during the coronavirus pandemic, have a 'natural' probability of becoming infected with the pathogen. Patients with prolonged hospital stays also have a higher risk of infection, especially those in facilities with similar patient profiles and diseases, as noted by Prof. Donker. He also underscored that P. aeruginosa infections are a growing global challenge. This pathogen thrives in aqueous environments and requires no special conditions to grow, making it a worldwide threat. Furthermore, Prof. Rieg considers a dual protection system for our project, as proposed by Prof. Tümmler, to be an effective and adaptive clinical solution. This would involve using ICE (Integrative and Conjugative Elements) in conjunction with our phage tail proteins on the outer membrane vesicles. The primary challenge is to develop the most effective therapy for as many infected patients as possible. An additional specification, such as the aforementioned ICE, is an optimal extension for CAPTURE. Prof. Rieg supports our idea of using an inhalation device, like our nebulizer, as a gentle treatment for people with lung infections. He is now our second expert, alongside Dr. Tümmler, to verify this application.

Implementation
This meeting helped us recognize two key aspects of our project. First, the importance of minimizing the hospital stay of high-risk patients with lung disease to reduce their risk of infection with P. aeruginosa. Second, as Prof. Rieg pointed out, our AMP could have a synergistic effect when used in combination with antibiotics, and the mechanism of action of AMPs helps prevent the development of drug resistance.

Outlook
Our conversation highlighted that P. aeruginosa is far more than a typical pathogen. While people with a healthy immune system can easily withstand this pathogen, it poses a significant threat to vulnerable patients. Those with compromised health, such as CF patients who are already dependent on artificial ventilation, are particularly at risk. Their intensive hospital treatments elevate them to high-risk status, making them more susceptible to severe and potentially fatal P. aeruginosa infections.

Key Takeaway

• To gain insights into the healthcare industry in Baden-Württemberg, we arranged further meetings with Special Interest Group (SIG) Health and Prof. Grundmann from the Freiburg University Hospital.

Purpose
To identify the needs of our project and to connect iGEM with the broader community in Freiburg, we decided to reach out to Dr. Hannes Dambach, head of the Healthcare Industry Cluster in Freiburg.

Contribution
Our first step was to present what iGEM is all about. Dr. Dambach was impressed by the young people's drive and ambition to tackle the world's problems with new and innovative ideas. That's why he recommended another meeting with the Special Interest Group (SIG) Health, a driver of the healthcare industry in Baden-Württemberg which also crosses borders into Switzerland and France, to create opportunities for exchange and networking with other research and business areas. He also recalled an article about a project from the first funding round, Initiative Patientensicherheit BW: Keine Chance für multiresistente Krankenhauskeime, about Prof. Dr. Hajo Grundmann from the University Hospital.

Implementation
To give iGEM more exposure, we extended our reach through various social media channels.

Outlook

• Meet up with the SIG-Health group
• Contact Prof. Dr. Hajo Grundmann