🐍 Toehold Switch Creator is an open source software based on python for the in silico design of the riboswitches called “Toehold switches”, we aimed to made an straightforward software in response to the dispersion of the bioinformatic tools used generally for designing these constructs, we originally made TSC for the development of our iGEM project: Diagnosgene a phytopathogenic detection system based on these biosensors.
After forking this repository you will need to perform the installation of the software dependencies, these installations are made with Anaconda, for a detailed explanation on the step-by-step installation guide for your specific OS visit our wiki information.
Once you have installed all the software dependencies, you can run Toehold Switch Creator with the following command. Remember to run it on the same directory you have all your input files.
python TH_Creator.pyIt is required 3 input files for the software to work: the first is a parameters file, containing the data PrimedRPA needs to compute to generate the RPA primers, secondly we require a FASTA file which is of crucial importance and contains the sequence to be detected and used by the program to compute the toehold switches as well as the primers, thirdly and lastly we need the energetic range wich will be used to calculate the suboptimal structures of the RNA toehold switches. On this repository you will find a template for the PrimedRPA parameters files as well as examples of each of these documents embedded within the Validations section, section, an example of the files could be as follows:
Tequilena_PrimedRPA_Parameters.txt
atequilena-sst1.fastaAfter computing the output file is an easy to employ .csv file that contains the toehold sequences generated for each primer pair, the sequences of the toeholds are arranged in score order, being the first structures the best switches assembled, however it is strongly recommended to employ a secondary structure visualization tool such as a NUPACK for a better understanding of the RNA structures, it is also recommended to select the structures with less suboptimal structures for being employed on the experimental validation.
The RPA primers are generated with PrimedRPA developed by Mathew Higgings, also the thermodynamic calculations employ ViennaRNA developed by Lorenz et al, the downloading of these dependencies are well described on the step by step guide provided on the Installation section.
This software was developed by Emiliano González Castañón, Juan Antonio Alfaro Almaguer and Emilio Fabian Ortiz.
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The following literature and softwares were utilised to design Toehold Switch Creator.
[1] Higgins, M., Ravenhall, M., Ward, D., Phelan, J., Ibrahim, A., Forrest, M. S., Clark, T. G., & Campino, S. (2019). PrimedRPA: primer design for recombinase polymerase amplification assays. Bioinformatics (Oxford, England), 35(4), 682–684. https://doi.org/10.1093/bioinformatics/bty701
[2] iGEM Thessaly. (2019). Dry lab. In silico analysis. https://2019.igem.org/Team:Thessaly/Model
[3] Higgins, M., Ravenhall, M., Ward, D., Phelan, J., Ibrahim, A., Forrest, M. S., Clark, T. G., & Campino, S. (2018). PrimedRPA: primer design for recombinase polymerase amplification assays. Bioinformatics, 35(4), 682–684. https://doi.org/10.1093/bioinformatics/bty701
[4] EPFL. (2017). Toehold Designer. Available at http://2017.igem.org/Team:EPFL/Software
[5] Green, A. A., Silver, P. A., Collins, J. J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 159(4), 925–939. https://doi.org/10.1016/j.cell.2014.10.002
[6] Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., Ferrante, T., Ma, D., Donghia, N., Fan, M., Daringer, N. M., Bosch, I., Dudley, D. M., O’Connor, D. H., Gehrke, L., & Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. https://doi.org/10.1016/j.cell.2016.04.059
[7] The Chinese Hong Kong University. (2017). Modeling: Designing Toehold Switch. Available at: http://2017.igem.org/Team:Hong_Kong-CUHK/Model