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Human Practices
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Thanks to advances in molecular biology and biochemistry, scientists have been able to consistently detect lower and lower concentration of molecules1, to the point that single molecules can be reliably recognized with methods such as polymerase chain reaction (PCR)2, fluorescence in situ hybridization (FISH)3 and enzyme-linked immunosorbent assays (ELISA)4. This has opened doors for synthetic biology to create better and more accurate diagnostic tests that use biomarkers like nucleic acids and proteins as targets5,6. Through such advances, the field of molecular diagnostics developed. Unfortunately, current standard methods require expensive equipment or trained personnel, which generally limits their usability to hospitals or laboratories. Recently, there has been a push to develop new tests that fuse the reliability of standard methods with affordable platforms such as lab-on-a-chip or paper strips to overcome this restrictions7-9. We wanted to help close this gap and set out to engineer a diagnosis principle for the detection of a wide array of targets that could be used without difficult-to-meet technical requirements.
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Make Munich (6th and 7th May 2017)
Make Munich is the south Germany«s biggest maker and do-it-yourself tech festival. This was a 2-day event where many innovative, technology loving makers had the opportunity to show their skills in their booths, in talks and in various workshops. This year there were a huge variety of booths and workshops from different categories. Also, the audience who visited the event included young kids, students, and seniors. This event was a perfect event for all kinds of audiences fascinated with technological advances.
We are very thankful to the Make Munich team for inviting us to this fascinating tech event. This was our very first public event where we did our first poster presentation and our first public interaction. We got many encouraging comments and ideas on how we could proceed in future. Plus, we also met many tech and synbio geeks who were very kind to give their views on our project development. We also had the opportunity to meet many startups which were based on synthetic biology as well. Plus, we also met some old iGEMers from different countries who gave us more tips on handling our iGEM project.
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IKOM Life Sciences (23rd May 2017)
IKOM is a voluntary student organization with 100 students from TUM, who organize career forums, scientific exhibitions and workshops annually. IKOM Life science is one of such event organized by IKOM where other students can meet different biotechnology and other related companies. It is highly recommended for all life science students and it is visited by students from all the universities in Munich.
This year's IKOM was really interesting with a high participation from different biotechnology, pharmaceutical and consulting companies. We met many companies from different parts of Munich and we also had the chance to talk to them personally. Some of those companies were interested in our work. Also, we had the chance to see what different companies can offer which helped us later to decide which sources to use in future.
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KLVHS youth meetup (3rd June 2017)
KVL organized the 3-days camping program with different workshops, presentations and activities for young students from schools and universities around Munich. The event was organized by a local group of youths which were involved in this organization. This is also a yearly event where the students camp there and are allowed to participate in all the events whenever they want as a part of this interaction. We were happy to give them some insights into our Project and give them an idea about synthetic biology and iGEM. Many were school students and had no idea about this field.
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Open House day LMU Biocenter (13th July 2017)
The open house day at the LMU Biocenter is an annual event that provides opportunity for everyone to come and experience the features of biological sciences. The Faculty of Biology at the LMU hosts this event for potential students, neighboring residents, and all interested citizens. In addition, the faculty also displays different research projects done at the Campus in Martinsried. This year, we had our own stall in the Biocenter along with other research groups from the LMU.
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Responsible research (20th July 2017):
The Responsible research was a one-day long symposium organized by the LMU Graduate School of the Life Sciences Grosshadern. There was a high participation of graduate students, postdocs, senior scientists and other interested students. Some of the topics that were discussed included some issues in research like: What is responsible research? How can we present data accurately and reproducibly? What exactly is plagiarism, and how can it be avoided? What do we need to know about open science? What is an Ombudsperson? These topics were especially directed for the next generation researchers. There were also keynote lectures from Professor Dr. Joachim Heberle (DFG Ombudsgremium for Science), FU Berlin, Professor Dr. Marcus Munaf (University of Bristol), Professor Dr. Debora Weber-Wulff (HTW Berlin). We also had breakout sessions on Avoiding Plagiarism, Animal Research, Clinical Trials, Experimental Design and Statistics, Image Analysis, Open Science. The program ended with a barbecue dinner and interaction.
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South African scouts talk (17th July 2017)
Some scouts from the Girls Guides South Africa came to visit our lab. It was a group composed of 5 instructors and 10 high school students. We did a poster presentation where we explained about the iGEM competition and synthetic biology. We also showed them the prototype of our device and explained them the concept and the inspiration behind our project. They were very interested in knowing more about working in the field of science and our motivation why we chose to take scientific careers. They asked us a lot of questions about antibiotic resistance and how antibiotic abuse endangers human health.
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Amgen meetup ( 4th August 2017)
We also presented our iGEM project CascAID to the 8 students of the Amgen Scholars program. Amgen Scholars Program provides undergraduate students from different fields with the opportunity to engage in a hands-on research experience at some of the world«s best institutions. In Europe, there are 5 hosts institutions who conduct the Amgen Scholars Program namely, ETH Zurich, Pasteur Institute, Karolinska Institute, LMU Munich, and the University of Cambridge.
The students who were part of the Amgen Scholars program in LMU Munich were invited to a special presentation with us. We answered their queries and gave them more information on synthetic biology and iGEM. Most of them were unaware of both and it was a great pleasure for us to be able to give them some insights into the iGEM world. Many were also highly motivated to hear that iGEM provides such a big platform for students interested in synthetic biology.
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Colorimetric read-outs
To couple CascAID with an easy read-out method we explored three colorimetric read-outs:
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AeBlue: The RNA strand in a specially designed RNA/DNA dimer is cut by Cas13a's collateral
activity. After digestion, the interaction between the two strands is too weak to hold the dimer and it
decays. We can then use the DNA-strand as template to translate the chromoprotein aeBlue.
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Diagram of aeBlue
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Intein-Extein: By binding TEV-protease with a RNA-linker we can use Cas13a's collateral activity
to regulate the protease's diffusion and use it to cleave a TEV tag separating the intein regions of a
modified chromophore. After the first cleavage, the intein segment excises itself13, bringing together the
halves of the chromophore. Only then is the chromophore functional and produces the colorimetric
read-out.
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Diagram of Intein-Extein
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Gold nanoparticles: Gold nanoparticles coated with short DNA sequences are held closely
together by a complementary linker RNA, which makes the solution intense blue14. Activated Cas13a cuts
the linker RNA, causing the nanoparticles to diffuse away from each other. This increase in distance
causes a color change to intense red.
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Gold nanoparticles
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Software
To help facilitate the design of crRNA, the sequences that give CascAID its specificity, we developed a
software tool that checks crRNA for unwanted secondary structures. This gives valuable insight on
whether the sequence is suited to use with Cas13a or whether some modifications are needed.
Together with Team Delft's software tool which designs the corresponding crRNA based on the target,
we collaborated to develop a powerful tool that suggests crRNA sequences and checks their usability
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References
- Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).
- Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.
- Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.
- Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.
- Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
- Slomovic, Shimyn, Keith Pardee, and James J. Collins. "Synthetic biology devices for in vitro and in vivo diagnostics." Proceedings of the National Academy of Sciences 112.47 (2015): 14429-14435.
- Tang, Ruihua, et al. "A fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection." Lab on a Chip 17.7 (2017): 1270-1279.
- Vashist, Sandeep Kumar, et al. "Emerging technologies for next-generation point-of-care testing." Trends in biotechnology 33.11 (2015): 692-705.
- Gubala, Vladimir, et al. "Point of care diagnostics: status and future." Analytical chemistry 84.2 (2011): 487-515.
- Abudayyeh, Omar O., et al. "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector." Science 353.6299 (2016): aaf5573.
- Gootenberg, Jonathan S., et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2." Science (2017): eaam9321.
- https://www.idtdna.com/pages/docs/technical-reports/in_vitro_nuclease_detectionD325FDB69855.pdf (retrieved: 13.10.17)
- Anraku, Yasuhiro, Ryuta Mizutani, and Yoshinori Satow. "Protein splicing: its discovery and structural insight into novel chemical mechanisms." IUBMB life 57.8 (2005): 563-574.
- Link, Stephan, and Mostafa A. El-Sayed. "Size and temperature dependence of the plasmon absorption of colloidal gold nanoparticles." The Journal of Physical Chemistry B 103.21 (1999): 4212-4217.
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