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<b> <center><p style="font-family: quicksand;font-size:200%;">RNA thermodynamic modeling: Designing Toehold Switch </p></center> </b> | <b> <center><p style="font-family: quicksand;font-size:200%;">RNA thermodynamic modeling: Designing Toehold Switch </p></center> </b> | ||
<p style="font-family: quicksand;font-size:130%;">Background </p> | <p style="font-family: quicksand;font-size:130%;">Background </p> | ||
− | <p | + | <p style="font-family: roboto;font-size:100%;"> |
− | 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 can have 970 possible switches. However, the performances of each possible switch will be different, since switches that target different region will have different thermodynamic characteristic and structure, which can affect the performance of the switch. Therefore, we modeled the thermodynamic and structure of our toehold switch during designing stage and simulate the expression of activated switch in silico. Our modelling helped us a lot in gaining insight. | + | 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 can have 970 possible switches. However, the performances of each possible switch will be different, since switches that target different region will have different thermodynamic characteristic and structure, which can affect the performance of the switch. Therefore, we modeled the thermodynamic and structure of our toehold switch during designing stage and simulate the expression of activated switch in silico. Our modelling helped us a lot in gaining insight. |
</p> | </p> | ||
</div> | </div> | ||
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<div class="column full_size"> | <div class="column full_size"> | ||
− | Toehold switch structure: | + | <p style="font-family: quicksand;font-size:130%;"> |
+ | Toehold switch structure: </p> | ||
<p><center><img src="https://static.igem.org/mediawiki/2017/d/dc/CUHK_toeholdstructure.jpg" width="50%" height="auto" class=" igem-logo"></center></p> | <p><center><img src="https://static.igem.org/mediawiki/2017/d/dc/CUHK_toeholdstructure.jpg" width="50%" height="auto" class=" igem-logo"></center></p> | ||
− | <p> | + | <p style="font-family: roboto;font-size:100%;"> |
We adopt the toehold switch design from the original paper. Our toehold switch contains 15nts “toehold domain”, 21nts stem-loop that contains a start codon and a RBS B0034 loop at that toop. A 21nts linker and mRFP reporter sequence is present downstream the toehold switch. The linker is used to separate the coding sequence in the toehold switch and the reporter to prevent interference of protein folding. | We adopt the toehold switch design from the original paper. Our toehold switch contains 15nts “toehold domain”, 21nts stem-loop that contains a start codon and a RBS B0034 loop at that toop. A 21nts linker and mRFP reporter sequence is present downstream the toehold switch. The linker is used to separate the coding sequence in the toehold switch and the reporter to prevent interference of protein folding. | ||
</p> | </p> | ||
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<div class="column full_size"> | <div class="column full_size"> | ||
− | < | + | <p style="font-family: quicksand;font-size:130%;">Assumptions and Definition:</p><br> |
− | <p><u><b>Assumption: Switch MFE ∝ leakage</b></u> | + | <p style="font-family: roboto;font-size:100%;"><u><b>Assumption: Switch MFE ∝ leakage</b></u> |
<br> | <br> | ||
<br> | <br> | ||
>> Switch MFE is the Gibbs free energy of a toehold switch folded in the most stable structure.<br> | >> Switch MFE is the Gibbs free energy of a toehold switch folded in the most stable structure.<br> | ||
>> Leakage is the level of reporter expression when switch is not activated by trigger.<br> | >> Leakage is the level of reporter expression when switch is not activated by trigger.<br> | ||
− | To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. Switch MFE reflects the difficulty for the toehold switch activation process. We assume that the more negative the Switch MFE, the harder for the activation to take place, and hence a lower leakage. | + | To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. Switch MFE reflects the difficulty for the toehold switch activation process. We assume that the more negative the Switch MFE, the harder for the activation to take place, and hence a lower leakage. </p> |
− | < | + | |
<br> | <br> | ||
<u><b>Assumption:∆ G RBS-Linker ∝ Dynamic range</b></u><br> | <u><b>Assumption:∆ G RBS-Linker ∝ Dynamic range</b></u><br> | ||
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<div class="column full_size"> | <div class="column full_size"> | ||
− | < | + | <p style="font-family: quicksand;font-size:130%;">Screening by our software</p><br> |
− | <p> | + | <p style="font-family: roboto;font-size:100%;"> |
To minimize the manpower on screening of the switches, we constructed an online toehold switch design program. 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 generated a list of possible Toehold Switch sequence according to their free energy using the embedded function of “Vienna RNA” (8). We ranked the ∆ G RBS- Linker as the most important parameter since it had already proven that it correlates with the dynamic range of switch. Below graph shows 394 possible H5 toehold switches generated by our software. We first chose the switches that with the highest ∆ G RBS- Linker (-3.8kcal/mol). Among those switches, we chose the 3 switches with low switch MFE and high MFE difference. | To minimize the manpower on screening of the switches, we constructed an online toehold switch design program. 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 generated a list of possible Toehold Switch sequence according to their free energy using the embedded function of “Vienna RNA” (8). We ranked the ∆ G RBS- Linker as the most important parameter since it had already proven that it correlates with the dynamic range of switch. Below graph shows 394 possible H5 toehold switches generated by our software. We first chose the switches that with the highest ∆ G RBS- Linker (-3.8kcal/mol). Among those switches, we chose the 3 switches with low switch MFE and high MFE difference. | ||
</p> | </p> |
Revision as of 12:34, 24 October 2017