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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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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. |
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, along with many other things, came from our supervisor Aurore. For the other targets that cover a wider spectrum of pathogens, Dawafuti was the main responsible for choosing and designing them when needed. The target sequences from viral origin were a kindly given by Killian Vogele, a PhD Student from Prof. Simmels group. Dawafuti and Julian experimented with the diverse RNA extraction and purification methods for detection with Cas13a.
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Interlab
Erika and Dawafuti took the interlab studies in their hands and, after being introduced by our supervisor Lukas to the machines, they two performed all cloning and experiments.
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RNA extraction
One of our two inital ideas for RNA extraction, Julian and Pattrick did multiple experiments trying to use beads to bind RNA. For our another option, Jorge experimented with the FINA extraction method as presented in McFall et al., 2015 and tried to adapt it for RNA extraction.
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Read-out
As we looked into colorimetric read-outs for CascAID, our supervisor Jeff helped us brainstorm and suggested two interesting options. One of them we dubbed AeBlue read-out and was explored by Christoph, who did part of the initial cloning, and Florian, who performed all main experiments. The other was the Intein-Extein read-out. The cloning and experimets for this read-out were done mainly by Max with help from Sven. Rob worked in the gold nanoparticles read-out with help from Killian Vogele, a PhD student in Prof. Simmels' group. Killian also kindly gave us some linker sequences necessary for this read-out.
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Lyophilization
Lyophilization, both of Cas13a and other components, was done by Rob, who reached out to Lady's name for guidance and equipment. With her support, Rob was able to use the machines at the Lady's institute to make the lyophilization experiments.
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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 Sven and Max developed "Heatbringer". Along the way, our supervisors Lukas and Benjamin gave valuable input and feedback. 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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Plate-reader experiments
Igor expend a lot of time and energy working with the plate-reader. He did most of the experiments with RNAase alert together with Dawafuti and Rob who tested the system with different targets and on paper. Our supervisor Aurore showed them how to use the plate reader and always provided great guidance.
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Human Pratices
Everyone on the team contributed to human practices either by organising 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. Among us, Teeradon played an important role in outreaching the community, as he wrote the survey questions and helped distribute them. 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. Benjamin acted as our supervisor for the wiki and provided us with helpful ideas and feedback. We'd like to thank Team Bristol for showing and teaching us the iGEM IDE which facilitated the work.
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Design, Presentation and Poster
As our art-guy, Benedikt was responsible for making for creating our amazing logo and banner, taking pictures, filming and styling our presentation. Him, together with Teeradon were in charge of designing the poster.
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Acknoledgments
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Even though they are not official members of the team, several people helped us develop our project. We'd 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 they patience, help and for showing us our way around the laboratory. Thanks to Jerome Lutz and the people at UnternehmerTUM for supporting us along the way with practical courses and ideas. Thanks to Lady's name for lending us their machine for the lyophilisation experiments. And many thanks to all our sponsors for their financial help and contributions.
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