Sequences for Amplifying MicroRNA
We designed and improved the template and helper sequences for amplifying miR-10b-5p, miR-30d-5p and miR-375 by TWJ-SDA and multistep-SDA and added them as parts. The template DNA and helper DNA we designed for TWJ-SDA specifically form a three-way junction complex with the target miRNA. This improves specificity and reduces false positive and false negative rates. Multistep-SDA also consists of a group of short single-stranded DNAs that are independent of target miRNAs.
We have registered a series of parts that can be used in combination with our software to designTWJ-SDA template and helper sequences specifically for amplifying any miRNA isothermally.
For more information about the sequences, see Parts.
CRISPR RNA (crRNA) Targeting N2 Gene Regions of SARS-CoV-2
We added crRNA targeting N2 gene regions of SARS-CoV-2 as parts. CRISPR-Cas3 used in our study is a technology developed by the Mashimo Laboratory, Division of Animal Genetics, the Institute of Medical Science, the University of Tokyo, for application to SARS-CoV-2 detection devices, and recognize N2 gene regions of SARS-CoV-2 1.
When the Cascade/crRNA complex recognizes and hybridizes with the target DNA strand, the Cas3 protein binds and nonspecifically breaks ssDNA around the target sequence. CRISPR-Cas3 is highly specific due to its long crRNA complement sequence of 32 bases 2.
Software
Our software was developed to support the applicability of our projects. Our software searches for miRNAs in body fluids, such as tear fluid and blood, and tells us if there are similar sequences to the miRNAs we set. In addition, our system can design nucleic acid sequences that are suitable for the amplification of the miRNA which users have set, so that the miRNA amplification and quantification system that we have developed can be used quickly. Automation of nucleic acid sequence design saves experiments and makes it easier to apply our system to various miRNAs.
You can use the software from the link below.
You can also refer to the Model, Software page for software algorithms and specific usage information.
For more information about the algorithm of our software, see Model.
For more information about how to use our software, see Software.
Manual
NUPACK
In our project, we used a software called NUPACK 3 many times to design nucleic acid sequences and to build models. We have created a manual that summarizes how to use NUPACK to help future iGEM teams use NUPACK when designing sequences and building models. The manual clearly explained how to use "Analysis" and its algorithms, which we mainly used.
The manual is as follows.
Molecular Dynamics Simulation
One of the most difficult things in this year's Dry Lab activity was performing Molecular Dynamics Simulation (MD Simulation). We leveraged our experience from this year's MD simulation to create an MD simulation manual to help future iGEM teams run MD simulations smoothly. The manual describes in detail how to use GROMACS 4 and GENESIS 5, 6, which we actually used this year.
The manual is as follows.
Education Material
Dry Education
We created educational materials to give students a hands-on experience of iGEM's Dry activity. In the first part of this material, students learn about gene circuits and can understand how the expression levels of proteins change depending on conditions by actually making graphs with their hands. In the second half, students can learn about ALDH2, a relatively familiar enzyme, and understand how differences in protein structure affect enzyme activity by drawing graphs.
Participants can use this material to experience Dry Lab activities, which might be difficult to imagine, without having to prepare any special equipment. This is thought to lead to increasing the resolution of iGEM for the general public.
The education material is as follows.
Brochure
We created a brochure to introduce this year's project of iGEM UTokyo.
Our project aims at the early detection of glaucoma. To inform readers about the symptoms of glaucoma, we intentionally blurred text and images in some parts of the brochure to reproduce the progression of visual field loss caused by glaucoma. The blurred brochures were distributed to the participants of our education activities. Those who received the brochure could also view the blur-free brochure by scanning the QR code on the brochure. At the bottom of the webpage with the blur-free pamphlet is a questionnaire about glaucoma, this year's project and the brochure. This will allow us to get feedback on our projects from people in society and will encourage two-way interaction.
The creation of such materials will be helpful for future iGEM teams dealing with ophthalmic diseases.
For more information about the brochure, see Education_Brochure & Questionnaire.
The brochures are as follows.
Brochure with blur
Brochure with no blur
Education Slides
The following puzzles have been created to help people who have never studied biology learn how the parts of a gene circuit function and how to design them. The puzzles, which allow you to organize conditions and to consider gene circuits in which the desired gene is expressed only when certain conditions are met, can be enjoyed regardless of whether or not you have knowledge of synthetic biology.
The slides we used are as follows.
References
Yoshimi, K., Takeshita, K., Yamayoshi, S., Shibumura, S., Yamauchi, Y., Yamamoto, M., Yotsuyanagi, H., Kawaoka, Y., & Mashimo, T. (2022). CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus.iScience 25, 103830, 1-13. https://doi.org/10.1016/j.isci.2022.103830
Yoshimi, K., Takeshita, K., Kodera, N., Shibumura, S., Yamauchi, Y., Omatsu, M., Umeda, K., Kunihiro, Y., Yamamoto, M., & Mashimo, T. (2022). Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3. Nature Communications, 13(4917). https://doi.org/10.1038/s41467-022-32618-0
Zadeh, J. N., Steenberg, C. D., Bois, J. S., Wolfe, B. R., Pierce, M. B., Khan, A. R., ... & Pierce, N. A. (2011). NUPACK: Analysis and design of nucleic acid systems. Journal of computational chemistry, 32(1), 170-173. https://doi.org/10.1002/jcc.21596
M.J. Abraham, T. Murtola, R. Schulz, S. Páll, J.C. Smith, B. Hess, and E. Lindahl. (2015). GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX, 1, 19–25. https://doi.org/10.1016/j.softx.2015.06.001
Kobayashi, C., Jung, J., Matsunaga, Y., Mori, T., Ando, T., Tamura, K., ... & Sugita, Y. (2017). GENESIS 1.1: A hybrid‐parallel molecular dynamics simulator with enhanced sampling algorithms on multiple computational platforms. Journal of Computational Chemistry, 38, 2193-2206. http://dx.doi.org/10.1002/jcc.24874
Jung, J., Mori, T., Kobayashi, C., Matsunaga, Y., Yoda, T., Feig, M., & Sugita, Y. (2015). GENESIS: a hybrid‐parallel and multi‐scale molecular dynamics simulator with enhanced sampling algorithms for biomolecular and cellular simulations. Wiley Interdisciplinary Reviews: Computational Molecular Science, 5(4), 310-323. https://doi.org/10.1002/wcms.1220
