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Attributions
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The iGEM competition demands a lot of work in terms of project planning, research, programming, fundraising, human practices and wet lab experiments. Because we are a team with a diverse background, we divided tasks to best match our member's experience and interests. Sometimes, that meant doing what we do best; other times, it meant learning something new. When difficulties arose, we turned to our supervisors for help and guidance. Here, we would like to credit our team members for the specific roles they played into making CascAID. Also, we would like to thank our supervisors and external help for
their important contributions to our project.
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Organization
As our team was composed of people with different lab-experience, there were some members who instructed the others in the use of lab equipment and methods. Our master students, Max, Ludwig and Sven teached the other team members and coordinated work distribution. Our supervisors Lukas and Aurore took over responsibility, supported the team in each point of this project and invested a lot of time in and outside the lab.
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Molecular cloning
Due to our background, most of us had experience in molecular cloning and thus almost every student
helped with it in some way. However, most of the work for the Cas13a expression in E. coli was done by
Ludwig and Christoph. The BioBricks were done by Rob and Max, who also cloned the constructs needed
for the Intein-Extein read-out. The parts for the aeBlue read-out were done by Florian and Christoph. All
the gene and primer design were done by the above-mentioned students and reviewed by our
supervisor Aurore.
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Protein purification
Our three Cas13a proteins: Lsh, Lwa and Lbu, were purified thanks to the hard work of Ludwig, Max,
Sven and Milica, who along with Aurore's feedback designed and cloned the parts necessary for the addition of the TEV-target-sequence. Dr. Sandra Sagredo, a Postdoc at Prof. Simmel´s group, introducted them to the Äkta Purification System.
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Targets
The original idea of using 16S rRNA from E. coli as a target for our initial experiments came from our
supervisor Aurore. Our pathogen targets were chosen and designed by Dawafuti. Kilian Vogele, a PhD
student from Prof. Simmel´s group, kindly provided us with the viral target sequences.
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RNA extraction
We tried several approaches for extracting RNA: Julian and Patrick did multiple experiments to use
beads to bind RNA; Jorge experimented with the FINA extraction method (McFall et al., 2015) and tried
to adapt it for RNA extraction; and Dawafuti and Julian also experimented with RNA extraction for different targets and purification methods for detection with Cas13a.
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Colorimetric read-outs
As we considered colorimetric readouts for CascAID, our supervisor Jeffery helped us brainstorm and suggested two interesting options: aeBlue and the Intein-Extein readout. The initial cloning for the first option was done by Christoph and Florian performed all main experiments. For the Intein-Extein system, Max, with help from Sven, did most of the cloning and experiments. Rob worked on the Gold Nanoparticles readout with help from Kilian Vogele, a PhD student in Prof. Simmel´s group. Kilian also kindly gave us some linker sequences necessary for testing this readout.
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Fluorescent read-out
Igor optimized the RNaseAlert system and cleavage assays with the in vitro targets and different Cas13a proteins in the plate reader. Dawafuti did experiments with the different targets and the Cas13a Lbu using the plate reader. She, together with Sven and Rob, tried different targets on paper. Our supervisor Lukas showed them how to use the plate reader and always provided great guidance.
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Lyophilization
The lyophilization of both Cas13a and other components, was done by Rob, who contacted Christine Braig from the IMETUM for guidance and equipment. With her support, Rob was able to use the “specific name of the
equipment” at the IMTETUM to make the lyophilization experiments.
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Interlab
Erika and Dawafuti were in charge of the InterLab Study. They received an introduction to the Plate
Reader and help setting the protocol for the measurements from our instructor Lukas. They both transformed the devices, did the measurement experiments, and processed the data.
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Hardware
Our hardware team consisted of four students: Katzi, Matthias, Sven and Max. Our fluorescence detector "Lightbringer" was conceived, designed and built by Katzi and Matthias, while the whole hardware team developed "Heatbringer", the thermocycler. Sven and Max worked on the initial design blueprints, both in design and execution; Matthias and Katzi then develop "Heatbringer" to its final version. Along the way, our supervisors Lukas, Benjamin and Michael gave valuable input and
feedback. Julia Müller und Marisa Götzfried, both PhD students in Prof. Simmel lab, helped our hardware team by giving tips regarding 3D-printing and circuit design. Jerome Lutz, from UnternehmerTUM, also helped us by giving us access to the awesome parts library in UnternehmerTUM's MakerSpace and offering us some courses there.
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Software
Jorge worked on the fluorescence detector's software under the instructions of Katzi, who designed the
algorithm. Sven and Max wrote most of the thermocycler's software, with support from Igor for the web interface.
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Human Practices and outreach
Everyone on the team contributed to human practices by organizing our attendance in one of the many
events we went to, representing us there, or reaching out scientists and the community to discuss our
project. For outreaching and acquiring sponsors, Matthias coordinated the communication between the different companies and our team. Thanks to Jerome Lutz from UnternehmerTUM for reaching out to for different events like the TECHFEST .
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Wiki
Our wiki was a joint effort mainly between Benedikt, who drew all the icons, logos, and designed most of the page, and Jorge, who did most of the coding with help from Milica and Igor. Our supervisor Benjamin provided us with helpful ideas and feedback. All our team members helped writing the wiki content. We would like to thank Team Bristol 2017 for creating and showing us the iGEM IDE which facilitated the work.
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Design, Presentation and Poster
As our main art-guy Benedikt created our amazing logo and banner, took pictures, filmed, and styled our presentation.
Together with Teeradon, he also was in charge of designing the poster.
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Social media
Our Facebook, Instagram, and Twitter accounts were managed mostly by Erika, Benedikt and Dawafuti.
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Acknowledgements
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Even though they are not official members of the team, several people helped us to develop our project. We would like to thank them for their help and support; without them CascAID wouldn't have been possible: Thanks to all the people in the Chair of Physics of Synthetic Biology Systems at the TUM for their patience, help and for showing us our way around the lab. Special thanks to Kilian Vogele (PhD Student), Aradhana Chopra (PhD Student), Julia Müller (PhD Student), Marisa Götzfried (PhD Student), and Dr. Sandra Sagredo (Postdoc). Thanks the people at UnternehmerTUM for supporting us along the way with practical courses and ideas. Thanks to Christine Braig for lending us her machine for the lyophilization experiments. And many thanks to all our sponsors for their financial help and contributions.
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