<p><h3><i>In silico</i> design of Influenza Toehold switches</h3></p>
<p><h3><i>In silico</i> design of Influenza Toehold switches</h3></p>
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According to Green <i>et al.</i>(1), the optimal length of RNA to be detected by a toehold switch is around 30 bp. In other words, a target RNA with 1000 bp in length will give 970 possible switches. However, the performances of each possible switches are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy of the RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model">RNA thermodynamics modelling page</a>). To minimize the manpower on screening of the switches, we constructed an <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> online toehold switch design program </a>. Apart from the basic thermodynamic parameters, it also screens for rare codons, stop codons and RFC illegal sites along the sequence. In addition, the built-in BLAST function also automatically screen for nonspecific region to avoid false positive detection. Ultimately, the program can sort a list of “best” Toehold Switch sequence according to their free energy using the embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (2). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.
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According to Green <i>et al.</i>(1), the optimal length of RNA to be detected by a toehold switch is around 30 bp. In other words, a target RNA with 1000 bp in length will give 970 possible switches. However, the performances of each possible switches are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy of the RNA (For more information, please visit <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model"> modelling page</a>). To minimize the manpower on screening of the switches, we constructed an <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> online toehold switch design program </a>. Apart from the basic thermodynamic parameters, it also screens for rare codons, stop codons and RFC illegal sites along the sequence. In addition, the built-in BLAST function also automatically screen for nonspecific region to avoid false positive detection. Ultimately, the program can sort a list of “best” Toehold Switch sequence according to their free energy using the embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (2). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.
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Firstly, they use luciferase as reporter gene. We think that luciferase is not feasible to be used for on-site diagnosis since the luciferase activity need to be measured by a reader. Secondly, we inputted the sequences of those oral cancer biomarkers into our program and found that a better switch can be designed by choosing another region for detection (see modelling page). Therefore, we improved their biobricks by using <i>in silico</i> design of switch and RFP as a reporter.
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Firstly, they use luciferase as reporter gene. We think that luciferase is not feasible to be used for on-site diagnosis since the luciferase activity need to be measured by a reader. Secondly, we inputted the sequences of those oral cancer biomarkers into our program and found that a better switch can be designed by choosing another region for detection (see <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Model"> modelling page</a>). Therefore, we improved their biobricks by using <i>in silico</i> design of switch and RFP as a reporter.