This year, we focused on two main aspects in our human practice engagement. We wanted to introduce synthetic biology to the public, making it more accessible. Therefore, we focused on Education and Public Engagement. Furthermore, we sought the cooperation with experts straight from the beginning of our project, integrating their input into every step of ALiVE.
Education and Public Engagement
Day of Initiatives at TUM
Open Day at the Biology Center of LMU
15x4 Munich - Bringing Science to the Public
iGEM@school
Alpine Meetup
Introduction to new students at TUM and LMU
Social Media takeover - German Network of Young Scientists e.V.
Straight from the start, we sought experts from academia and industry to tailor our platform according to their needs for seamless integration into pre-existing workflows. To assess the public reception of our project, we presented ALiVE at a science communication event. Public safety concerns were consequently discussed with an expert on ethics and safety in medicine. Finally, to design our platform more user-oriented, we approached stakeholders to discuss ALiVE's potential to solve current problems in live-cell monitoring and differentiation of iPS cells.
April
Exosome Constructs
Prof. Dr. Martin Fussenegger
Prof. Dr. Martin Fussenegger has already established exosomes as a cargo container. After intensive literature research, we reached out to Prof. Fussenegger to inquire whether we could use his basic constructs.
Integration
Aquired our RNA adpater protein L7Ae
Aquired the exosome's base protein CD63
May
VLP Constructs
Christoph Gruber
As one of our scientific advisors, Christoph Gruber discussed our project idea with us early on. From him we recieved our Virus-like-Particle (VLP) base construct Gag as well as our second RNA adapter protein.
Integration
Aquired our second RNA adpater protein MCP
Aquired the VLP's base protein Gag
Safety aspects of VLPs
Virus Expert
After we had aquired our base parts, we had to decide how to use them. Especially for the viral parts, we were concerned about ethical aspects of our project, as using viral components in a diagnostic project needs careful evaluation. Therefore, we sought out an HIV-expert, with whom we discussed ethical concerns and safety aspects of our project, especially the VLP aspects.
Feedback
The expert discouraged us from using VSV-G, another viral protein, due to its increased transfection efficiency.1
She emphasized the necessity to develop assays that could monitor the transfection efficiency of our VLPs.
Integration
We decided not to use VSV-G as a VLP-secretion enhancer due to its safety concerns.1
We developed a luminescence- and qPCR-based infectivity, or "collateral transfection" assay.
July
Exosome Purification
Prof. Dr. Franz Hagn
During the development of our purification protocols, we contacted Prof. Dr. Franz Hagn, a membrane protein expert. Our goal was to engineer CD63, a transmembrane protein, by inserting a His-tag into the outer loops. As the structure of CD63 is not yet resolved, we used the structure of a similar protein, CD81, to model the His-tag. Here, Prof. Hagns insight in membrane protein structures was very helpful.
Feedback
Design criteria for the engineered His-tag
Input on the optimal position for a His-tag within the extracellular loop of the protein
Integration
We designed three His-tagged protein versions, from which we tested two successfully
Prof. Hagns input helped us to increase exosome purification significantly
Exosomes can now be purified over affinity purification for the first time due to our novel construct, making it our best basic part.
September
Exosome Diagnostics
Dr. Daniel Enderle
To evaluate the diagnostic potential of our project, we presented our concept and data to Dr. Daniel Enderle, an exosome diagnostics expert working in the industry. With him we discussed our exosome construct design, as well as our assay design and procedures. Due to his experience with qPCR, the feedback here was especially valuable.
Construct Design
Evaluation of construct positioning
Approval of HiBiT as an analytic tool
Dr. Enderle explained that exosomes represent the cytosols content, further encouraging us that exosome-based transcriptomics could be possible
Plasmid Contaminations
Dr. Enderle mentioned, that high noRT values are often due to plasmid contamination
Description of how best to cleanse the area where the qPCR is prepared
Primer Design
Insight in the impact of primer design on qPCR results
Tips on how to design qPCR primers
qPCR Techniques
Threshold set to fixed instead of auto-adjusted
setup of external balance line
Feedback
Approved the exosome construct design
Advice on how to avoid plasmid contaminations
Design criteria for qPCR primer
Suggestions regarding qPCR evaluation
Integration
Bleech cleansing of parts of the laboratory and installation of "plasmid-free" zones
Design of new qPCR primers, implementing Dr. Enderle's advice
Change from adaptable to steady qPCR threshhold and balance line modification
Public Reception
15x4 Science Communication Event
Human practices does not only mean to speak with experts and scientists, but also to interact with the public in general. Therefore, our team presented ALiVE in front of a mostly non-scientific audience of over 200 people. Afterward, we took questions, on and off the stage to understand the public's perception of our project and synthetic biology in general, especially when used as a diagnostic tool.
Feedback
Using genetically modified organisms (GMOs) for therapeutics and diagnostics is a topic the public is concerned about.
The audience was very interested in the genetic modification and its procedure.
A common concern was the potential harm ALiVE could cause due to the vesicles uncontrolled presence in patients.
Integration
Based on the audience's feedback we reevaluated our presentation and our application examples to make ALiVE easily understandable without being intimidating.
The public outreach reaffirmed the importance of our collateral transfection assay as well as cytotoxicity monitoring, which we integrated into our course of experiments.
October
Ethics and Safety
Prof. Dr. med. Gerrit Hohendorf
After the science communication event, we wanted to discuss the collected concerns with an expert. Therefore, we sought to discuss ethical concerns and safety aspects of our project.
Feedback
ALiVE as a tool for fundamental research holds no strong concerns.
With ALiVE serious medical interventions such as biopsies for the analysis of transplanted cells can be prevented.
As a diagnostic tool for medical pruposes, the obstacles are higher. But due to therapies like CAR-T cell therapies, the clinical use of GMOs is not impossible.
There is always a higher risk of developing cancer when bringing mutated cells in an organism.
Integration
We designed our in silico model to predict the efficiency of ALiVE.
We talked to Prof. Dr. med. Martin Hildebrandt as a T - cell expert about the application of ALiVE in CAR-T cells.
Political obstacles regarding ALiVE as a diagnostic tool were subsequently discussed with our entrepreneurial advisory board.
ALiVEs potential to monitor cell differentiation over cell cycle reporters was discussed with experts.
With careful consideration and integration of our human practices outreach, we successfully developed a working proof of principle. With the feedback of the virus expert, Prof. Dr. Hohendorf and the public, we formed ALiVE by considerating ethical and safety aspects. The scientific input was continuoulsy integrated to design and realize the project in the best way possible. Due to the input of Prof. Dr. Hagn, we were able to develop a novel exosome purification construct, which can be used from now on by anyone.
Due to the positive feedback and our working proof of principle, we established an end-user orientated and start-up experienced advisory board to realize ALiVE's market potential.
