(8 intermediate revisions by 2 users not shown) | |||
Line 52: | Line 52: | ||
<div class="column full_size"> | <div class="column full_size"> | ||
<p style="font-family: quicksand;font-size:150%;">RNA thermodynamic Modelling</p> | <p style="font-family: quicksand;font-size:150%;">RNA thermodynamic Modelling</p> | ||
+ | |||
+ | <center><img src="https://static.igem.org/mediawiki/2017/9/90/Toehold_Switch_Model.jpeg" width="100%" height="auto" class=" igem-logo"></center> | ||
+ | <p style="font-family: roboto;font-size:115%;"> | ||
+ | |||
<p style="font-family: roboto;font-size:125%;color:blue"><b>Assumption 1: Switch MFE (Minimum Free Energy) correlates with the expression leakage</p></b> | <p style="font-family: roboto;font-size:125%;color:blue"><b>Assumption 1: Switch MFE (Minimum Free Energy) correlates with the expression leakage</p></b> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
Line 65: | Line 69: | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
● 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.<br> | ● 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.<br> | ||
− | ● The duplex expression is the reporter expression of the switch-trigger | + | ● The duplex expression is the reporter expression of the switch-trigger duplex RNA. |
<br> | <br> | ||
− | After the switch RNA hairpin is unwound after binding to the trigger RNA, a switch-trigger | + | After the switch RNA hairpin is unwound after binding to the trigger RNA, a switch-trigger duplex RNA would be formed. The RBS-linker region of the MFE structure of this duplex 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. |
<br><br> | <br><br> | ||
Line 101: | Line 105: | ||
<div class="column full_size"> | <div class="column full_size"> | ||
− | <p style="font-family: quicksand;font-size:150%;"> | + | <p style="font-family: quicksand;font-size:150%;">Initial screening using our software</p> |
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | 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 other factors(please visit our <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> software page </a>. Ultimately, the program generated a list of possible toehold switch sequences according to many different free energy parameters using the <a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/barriers.cgi">ViennaRNA package</a> [2]. The graph below shows 394 possible H5 toehold switches generated by our software. The assumptions motioned earlier stated 3 very important parameters for the selection of switch candidates with the greatest possible performances: Switch MFE, ΔG<sub>RBS-linker</sub>, and ΔMFE. We applied these parameters to our switch selection process: We first chose the switches that with the highest ΔG<sub>RBS-linker</sub> (-3.8 kcal/mol). Among those switches, we chose the 3 switches with the lowest switch MFE and the highest ΔMFE. | + | 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 other factors(please visit our <a href="https://2017.igem.org/Team:Hong_Kong-CUHK/Software"> software page</a>. Ultimately, the program generated a list of possible toehold switch sequences according to many different free energy parameters using the <a href="http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/barriers.cgi">ViennaRNA package</a> [2]. The graph below shows 394 possible H5 toehold switches generated by our software. The assumptions motioned earlier stated 3 very important parameters for the selection of switch candidates with the greatest possible performances: Switch MFE, ΔG<sub>RBS-linker</sub>, and ΔMFE. We applied these parameters to our switch selection process: We first chose the switches that with the highest ΔG<sub>RBS-linker</sub> (-3.8 kcal/mol). Among those switches, we chose the 3 switches with the lowest switch MFE and the highest ΔMFE. |
</p> | </p> | ||
− | |||
− | <p><center><img src="https://static.igem.org/mediawiki/2017/7/78/CUHK_softwarecandidate.jpg" width=" | + | <p><center><img src="https://static.igem.org/mediawiki/2017/7/78/CUHK_softwarecandidate.jpg" width="40%" height="auto" ></center></p> |
<br><br><br> | <br><br><br> | ||
Line 318: | Line 321: | ||
<div class="column full_size"> | <div class="column full_size"> | ||
<p style="font-family: quicksand;font-size:150%;">Suboptimal Structure Modelling</p> | <p style="font-family: quicksand;font-size:150%;">Suboptimal Structure Modelling</p> | ||
− | <p style="font-family: roboto;font-size:125%;" | + | <p style="font-family: roboto;font-size:125%;color:blue"><b>Assumption 4: Accessibility of toehold domain correlates with the performance of switch</p></u> |
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
● The toehold domain (first 15 nts) of the switch RNA is crucial for the binding of the trigger RNA to the switch RNA to initiate the switch-unwinding process: | ● The toehold domain (first 15 nts) of the switch RNA is crucial for the binding of the trigger RNA to the switch RNA to initiate the switch-unwinding process: | ||
− | |||
− | |||
Toehold domain must have minimal paired bases in the switch RNA to ensure the successful binding of this domain with the complementary sequence in the trigger RNA, which allows the unwinding of the switch RNA and permits translation of the reporter protein, RFP, to occur. </p> | Toehold domain must have minimal paired bases in the switch RNA to ensure the successful binding of this domain with the complementary sequence in the trigger RNA, which allows the unwinding of the switch RNA and permits translation of the reporter protein, RFP, to occur. </p> | ||
Line 506: | Line 507: | ||
<br> | <br> | ||
− | We checked the status of the toehold domain for each suboptimal structure from the ViennaRNA web server output [2] to see if it was open or closed. In the table, the structures with an open toehold domain are shown in < | + | We checked the status of the toehold domain for each suboptimal structure from the ViennaRNA web server output [2] to see if it was open or closed. In the table, the structures with an open toehold domain are shown in <font color="green">green</font>, and ones with a closed toehold domain are shown in <font color="red">red</font>. |
<br> | <br> | ||
For example, for H5-3 switch: | For example, for H5-3 switch: | ||
Line 516: | Line 517: | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | From the table above, we observed that only 3 out of 15 of our predictions were incorrect, showing promising accuracy of this prediction method. None of the 3 incorrect predictions were false negatives, indicating that the suboptimal structure prediction method should be mostly applied to filtering out switch sequences with low predicted performances.</p> | + | From the table above, we observed that only <em>3 out of 15 of</em> our predictions were incorrect, showing promising accuracy of this prediction method. None of the 3 incorrect predictions were false negatives, indicating that <strong>the suboptimal structure prediction method should be mostly applied to filtering out switch sequences with low predicted performances</strong>.</p> |
<br> | <br> | ||
Line 522: | Line 523: | ||
<div class="column full_size"> | <div class="column full_size"> | ||
− | <p style="font-family: quicksand;font-size:150%;"> | + | <p style="font-family: quicksand;font-size:150%;">Experimental verification of the thermodynamic mode</p> |
<p style="font-family: roboto;font-size:125%;"><u><b> Correlation of switch MFE (Minimum Free Energy) and expression leakage </p></b></u> | <p style="font-family: roboto;font-size:125%;"><u><b> Correlation of switch MFE (Minimum Free Energy) and expression leakage </p></b></u> | ||
<img src="https://static.igem.org/mediawiki/2017/1/1e/CUHK_SMFE.jpg" width="40%" height="auto" class=" igem-logo"> | <img src="https://static.igem.org/mediawiki/2017/1/1e/CUHK_SMFE.jpg" width="40%" height="auto" class=" igem-logo"> | ||
Line 532: | Line 533: | ||
<img src="https://static.igem.org/mediawiki/2017/d/d9/CUHK_ScMFE2.jpg" width="40%" height="auto" class=" igem-logo"> | <img src="https://static.igem.org/mediawiki/2017/d/d9/CUHK_ScMFE2.jpg" width="40%" height="auto" class=" igem-logo"> | ||
<br> | <br> | ||
− | Although the switch MFE correlates with the expression leakage, this plot showed that the switches with lower MFE also have lower RFP signal in the presence of trigger. This suggests that a low MFE | + | Although the switch MFE correlates with the expression leakage, this plot showed that the switches with lower MFE also have lower RFP signal in the presence of trigger. This suggests that a low switch MFE could also be a hindrance to detection that it increases the energy input to the system (activation energy), so it takes more energy for switch-trigger duplex formation to occur. This could lower the expression level of the switch-trigger duplex. |
<br> | <br> | ||
<br> | <br> | ||
Line 538: | Line 539: | ||
<img src="https://static.igem.org/mediawiki/2017/c/c1/CUHK_MFED.jpg" width="40%" height="auto" class=" igem-logo"> | <img src="https://static.igem.org/mediawiki/2017/c/c1/CUHK_MFED.jpg" width="40%" height="auto" class=" igem-logo"> | ||
<br> | <br> | ||
− | We initially thought that the larger the MFE difference between the sum of the MFE of switch and trigger and that of the switch-trigger duplex, the more favourable the duplex structure will be. However, there seems to be a weak positive correlation between the expression level of the switch | + | We initially thought that the larger the MFE difference between the sum of the MFE of switch and trigger and that of the switch-trigger duplex, the more favourable the duplex structure will be. However, there seems to be a weak positive correlation between the expression level of the switch-trigger duplex and the MFE difference of a switch. This could be because that a low MFE difference could indicate a more similar conformation of the switch/trigger RNA to the switch-trigger duplex RNA. This could lead to less conformational changes in the duplex formation process, making the process faster. Thus, a small MFE difference could lead to an increased expression level of the switch-trigger duplex. |
<br> | <br> | ||
<br> | <br> | ||
Line 544: | Line 545: | ||
<img src="https://static.igem.org/mediawiki/2017/3/33/CUHK_bp.jpg" width="40%" height="auto" class=" igem-logo"> | <img src="https://static.igem.org/mediawiki/2017/3/33/CUHK_bp.jpg" width="40%" height="auto" class=" igem-logo"> | ||
<br> | <br> | ||
− | The plot shows some kind of negative correlation between RFP signal in the presence of trigger and the number of base pairs in the toehold domain. Some exceptions were observed, they are N1-3, N9-3, N9-1. H5-3 and N1-2. This supports the | + | The plot shows some kind of negative correlation between RFP signal in the presence of trigger and the number of base pairs in the toehold domain. Some exceptions were observed, they are N1-3, N9-3, N9-1. H5-3 and N1-2. This supports the assumption in the suboptimal structure modelling that an increasing number of unpaired bases in the toehold domain promotes initial binding of a trigger RNA to the corresponding switch RNA. |
Latest revision as of 04:01, 2 November 2017