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<p style="font-family: quicksand;font-size:150%;">Background </p> | <p style="font-family: quicksand;font-size:150%;">Background </p> | ||
<p style="font-family: roboto;font-size:115%;"> | <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 can have 970 possible switches | + | According to Green et al. (2014)[1], the optimal length of RNA to be detected by a toehold switch is around 30 bp (complementary region at below figure). In other words, a target RNA with 1000 bp in length can have 970 possible switches with different performance, which is governed by their structures and thermodynamic parameters. We aim at providing a workflow and platform of modeling to help users design the switches by reducing the manual processing and increasing the hit-rate of finding a good switch. |
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− | + | We adopt the toehold switch design from the original paper(below figure). Our toehold switch contains 15 nts “toehold domain”, 18nts stem and a loop that contains the RBS B0034. A 21 nts linker sequence plus an 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. | |
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<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> | ||
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</p> | </p> | ||
</div> | </div> | ||
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<p style="font-family: roboto;font-size:125%;"><u><b>Assumption: Switch MFE (Minimum Free Energy) correlates with the expression leakage</p></b></u> | <p style="font-family: roboto;font-size:125%;"><u><b>Assumption: Switch MFE (Minimum Free Energy) correlates with the expression leakage</p></b></u> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | + | ● Switch MFE is the minimum Gibbs free energy that a toehold switch could have among all the possible structures.<br> | |
− | + | ● Expression leakage is a phenomenon where the reporter (i.e. Red Fluorescent Protein in our project) is expressed in the absence of trigger RNA. The level of leakage can be measured as: | |
− | To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. The Switch MFE reflects the difficulty for the toehold switch unwinding process. We assume that the more negative the Switch MFE, the harder for the unwinding to take place, and hence a lower leakage.</p> | + | |
+ | <p><center><img src="https://static.igem.org/mediawiki/2017/3/32/CUHK_leakage.png" width="25%" height="auto" class=" igem-logo"></center></p> | ||
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+ | To activate toehold switch, an amount of energy is needed to open the toehold switch hairpin. The Switch MFE reflects the difficulty for the toehold switch unwinding process. We assume that the more negative the Switch MFE, the harder for the unwinding to take place, and hence a lower leakage. | ||
+ | </p> | ||
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− | <u><b><p style="font-family: roboto;font-size:125%;">Assumption: ΔG<sub>RBS- | + | <u><b><p style="font-family: roboto;font-size:125%;">Assumption: ΔG<sub>RBS-Linker</sub> correlates with the duplex expression</p></b></u> |
− | + | ● ΔG<sub>RBS-Linker</sub> is the Gibbs free energy of the RNA sequence starting from the RBS to the linker in the switch-trigger duplex (Figure).<br> | |
− | + | ● The duplex expression is the reporter expression of the switch-trigger dimer RNA. | |
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− | After the switch RNA hairpin is unwound after binding to the trigger RNA, a switch-trigger dimer RNA would be formed. The RBS-linker region of the MFE structure of this dimer RNA should have minimal base pairs. This makes it easier to unwind the RNA for this region, allowing ribosomes to bind to the RBS and move along the RNA for translation of the RFP reporter gene to occur. ΔG<sub>RBS- | + | After the switch RNA hairpin is unwound after binding to the trigger RNA, a switch-trigger dimer RNA would be formed. The RBS-linker region of the MFE structure of this dimer RNA should have minimal base pairs. This makes it easier to unwind the RNA for this region, allowing ribosomes to bind to the RBS and move along the RNA for translation of the RFP reporter gene to occur. ΔG<sub>RBS-Linker</sub> reflects the difficulty for the unwinding process of the RBS-linker region. It is assumed that the more negative the ΔG<sub>RBS-Linker</sub> , the harder it is for the unwinding to take place, leading to lower translation rates. Thus, the duplex expression would be reduced. |
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<u><b><p style="font-family: roboto;font-size:125%;">Assumption: ΔMFE correlates with the duplex expression</b></u></p> | <u><b><p style="font-family: roboto;font-size:125%;">Assumption: ΔMFE correlates with the duplex expression</b></u></p> | ||
− | + | ● ΔMFE is defined as MFE of the switch-trigger duplex RNA minus (switch RNA MFE + trigger RNA MFE). | |
− | Since ΔMFE = –RTlnK, where:<br> | + | <br> |
− | R=gas constant<br> | + | Since ΔMFE = –RTlnK, where: |
− | T=temperature<br> | + | <br> |
− | K=equilibrium constant | + | R=gas constant |
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− | Therefore, we assume that the | + | T=temperature |
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+ | K=equilibrium constant | ||
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+ | Therefore, we assume that the more negative the MFE difference is, the higher the switch-trigger duplex RNA concentration compared to that of the switch RNA when in equilibrium. | ||
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+ | <center>Switch RNA+Trigger RNA↔Duplex RNA</center> | ||
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Consequently, increased equilibrium concentrations of the switch-trigger duplex RNA would provide an increased number of active mRNAs for the translation of the reporter RFP. | Consequently, increased equilibrium concentrations of the switch-trigger duplex RNA would provide an increased number of active mRNAs for the translation of the reporter RFP. | ||
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+ | (THE toehold domain base pairing modelling would be covered by the switch RNA suboptimal structure modelling part) | ||
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</p> | </p> | ||
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Revision as of 07:10, 1 November 2017