<center> <h1>Overview of experiment</h1> </center>
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<center> <h1>Overview of experiments</h1> </center>
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<p style="font-family: roboto;font-size:115%;">In our project, we first designed toehold switches the detect H7N9 and H5N1 virus in silico based on our modelling. We then constructed the plasmids that express switches and triggers by DNA synthesis and standard cloning method. Validation of the toehold switches was performed by co-expressing the switch and trigger plasmids in E. coli and in cell free system. Meanwhile, we also constructed our toehold switches and trigger cloning tool to allow convenient construction of switch and trigger expressing plasmid. Using the cloning tool, we tried to improve one existing toehold switch in the Registry. We also characterized two reporter protein (mRFP and amaJlime) in the registry to facilitate our project.
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<ol type="1">
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<p style="font-family: roboto;font-size:115%;">
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<li><i>In silico</i> designed toehold switches to detect H7N9 and H5N1 viruses based on our modelling.</li>
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<li>Constructed plasmids containing switches or triggers by DNA synthesis and standard cloning method.</li>
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<li>Validated the toehold switches by co-expressing the switch and trigger plasmids in <i>E. coli</i> and cell free system.</li>
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<li>Constructed cloning tools for toehold switch and trigger to allow convenient replacement of switch and trigger in the plasmids.</li>
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<li>Improved one existing toehold switch in the Registry.</li>
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<li>Characterized two reporter proteins (mRFP and amaJlime) in the Registry.
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<p><h3>In silico design of Influenza Toehold switches</h3></p>
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<p><h3><i>In silico</i> design of Influenza Toehold switches</h3></p>
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<p style="font-family: roboto;font-size:115%;">
According to Green et al., 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 switch are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy (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 basic thermodynamic parameters, it also screens for rare codon, stop codon 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> (8). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.
According to Green et al., 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 switch are different, since the performance is governed by serval parameters in the target region, such as the minimum free energy (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 basic thermodynamic parameters, it also screens for rare codon, stop codon 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> (8). The program facilitates the construction of toehold switch by providing a user-friendly interface with novel screening function.