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</div> | </div> | ||
+ | <tr class="lastRow"><td colspan=6 align=center valign=center> | ||
+ | <table class="myTable" width=100%> | ||
+ | <th class="leftAligned">rate constant</th> | ||
+ | <th class=”leftAligned”>value</th> | ||
+ | <th class="leftAligned">reference or rationale</th> | ||
+ | <tr> | ||
+ | <td class="leftAligned">k<sub>cr</sub></td> | ||
+ | <td class="leftAligned">1 [1/min]</td> | ||
+ | <td class="leftAligned">Mekler et al. (2016) Nucleic Acids Resarch</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="leftAligned">k<sub>t</sub></td> | ||
+ | <td class="leftAligned">0.001 [1/min]</td> | ||
+ | <td class="leftAligned">Estimated to be slow in comparison to k<sub>cr</sub></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="leftAligned">k<sub>col</sub></td> | ||
+ | <td class="leftAligned">10 [1/min]</td> | ||
+ | <td class="leftAligned">Estimated to be fast in comparison to k<sub>cr</sub> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="leftAligned">k<sub>RT-RPA-Tx</sub></td> | ||
+ | <td class="leftAligned">0.4 [1/min]</td> | ||
+ | <td class="leftAligned">Estimated from RPA-Tx amplification experiments</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="leftAligned">K<sub>M</sub></td> | ||
+ | <td class="leftAligned">500 [nM]</td> | ||
+ | <td class="leftAligned">Weitz et al. (2014) Nature Chemistry</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
<p> | <p> | ||
As shown in <b>Figure 1</b>, our simulations are able to reproduce the behavior observed experimentally. | As shown in <b>Figure 1</b>, our simulations are able to reproduce the behavior observed experimentally. | ||
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<img alt="LightbringerReal" src="https://static.igem.org/mediawiki/2017/8/87/T--Munich--ModellingPagePicture_kinetics_Cas13a_only.png" width="700"> | <img alt="LightbringerReal" src="https://static.igem.org/mediawiki/2017/8/87/T--Munich--ModellingPagePicture_kinetics_Cas13a_only.png" width="700"> | ||
<p> | <p> | ||
− | Figure 1: Kinetics of Cas13a using 1 nM and 10 nM at different target concentrations. | + | Figure 1: Kinetics of Cas13a using 1 nM Cas13a and 10 nM crRNA at different target concentrations. |
</p> | </p> | ||
</div> | </div> | ||
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<img alt="LightbringerReal" src="https://static.igem.org/mediawiki/2017/b/b8/T--Munich--ModellingPagePicture_CascAID.png" width="600"> | <img alt="LightbringerReal" src="https://static.igem.org/mediawiki/2017/b/b8/T--Munich--ModellingPagePicture_CascAID.png" width="600"> | ||
<p> | <p> | ||
− | Figure 2: Estimated detection limit determined for the Cas13a system using | + | Figure 2: Estimated detection limit determined for the Cas13a system using 1 nM Cas13a and 10 nM crRNA. |
</p> | </p> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | <h3>Improved Reaction Cascade</h3> | ||
+ | <p> | ||
+ | |||
+ | |||
+ | In collaboration with our wetlab team we developed a reaction cascade for sample pre-amplification by coupling reverse transcription to isothermal recombinase polymerase amplification and transcription (RT-RPA-TX), resulting in auto-catalysis of target RNA <b>(Figure 3)</b>.</p> | ||
<div class="captionPicture"> | <div class="captionPicture"> | ||
− | <img | + | <img width=600 src="https://static.igem.org/mediawiki/2017/d/dc/T--Munich--ModellingPagePicture_RT-RPA-TX_scheme.svg" alt="RT-RPA-TX_scheme"> |
<p> | <p> | ||
− | Figure 3: | + | Figure 3: Scheme for the RT-RPA-TX amplification system. |
</p> | </p> | ||
</div> | </div> | ||
− | + | <p> | |
− | + | In order to compare the detection limit of the Cas13a system alone with the detection limit of the amplified the reaction cascade, we expanded our model, assuming exponential amplification of the target RNA. As the amplification reaction saturates due to a depletion of resources, the amplification stops as soon as the target RNA level reaches an upper limit of 1000 nM <b>(Figure 4)</b>. | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <p> | + | |
− | In order to compare the detection limit of the Cas13a | + | |
− | + | ||
− | + | ||
</p> | </p> | ||
+ | |||
<div class="captionPicture"> | <div class="captionPicture"> | ||
<img width=800 align=center valign=center src="https://static.igem.org/mediawiki/2017/1/16/T--Munich--ModellingPagePicture_scheme2.png" alt="RT-RPA-TX"> | <img width=800 align=center valign=center src="https://static.igem.org/mediawiki/2017/1/16/T--Munich--ModellingPagePicture_scheme2.png" alt="RT-RPA-TX"> | ||
<p> | <p> | ||
− | Figure | + | Figure 4: Schematic representation of the target RNA amplification during the estimation of the detection limit using the reaction cascade. |
+ | </p> | ||
+ | </div> | ||
+ | <p> | ||
+ | The kinetics for the amplfication cascade coupled to Cas13a based detection are shown in <b>Figure 5</b>. Strikingly, the start of the reaction seems to be determined by the amplificaiton reaciton, while the consecutive phase is limited by the rate of Cas13a mediated cleavage. | ||
+ | As shown in <b>Figure 2</b>, the detection limit of the reaction cascade decreases by approximately three orders of magnitude. These simulations led us to implement our pre-amplification cascade into our CascAID system. | ||
</p> | </p> | ||
− | |||
<div class="captionPicture"> | <div class="captionPicture"> | ||
− | <img | + | <img alt="LightbringerReal" src="https://static.igem.org/mediawiki/2017/1/14/T--Munich--ModellingPagePicture_kinetics_Cas13a.png" width="600"> |
<p> | <p> | ||
− | Figure | + | Figure 5: Kinetics of the Cas13a systemusing 1 nM Cas13a and 10 nM crRNA at different target concentrations using the reaction cascade. |
</p> | </p> | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | |||
+ | |||
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in order to release enough RNA for downstream amplification. For this, we constructed a very simplistic | in order to release enough RNA for downstream amplification. For this, we constructed a very simplistic | ||
model for bacterial cell lysis. In this, we estimated the rate constants for cell lysis by common colony PCR | model for bacterial cell lysis. In this, we estimated the rate constants for cell lysis by common colony PCR | ||
− | protocols which use a 10 minute lysis step at 95 °C for thermolysis. Thus, we considered a half- | + | protocols which use a 10 minute lysis step at 95 °C for thermolysis. Thus, we considered a half-life of bacteria |
of 2 minutes at 95 °C. This would result in a lysis efficiency of 96.875%. Starting from this estimation, | of 2 minutes at 95 °C. This would result in a lysis efficiency of 96.875%. Starting from this estimation, | ||
− | we considered the rate constant of lysis and thus the half- | + | we considered the rate constant of lysis and thus the half-life using Arrhenius equation as commonly done in the literature: |
</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/b/b0/T--Munich--Model_Equation_1.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/b/b0/T--Munich--Model_Equation_1.png"><span>(7)</span></div> |
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/3/30/T--Munich--Model_Equation_2.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/3/30/T--Munich--Model_Equation_2.png"><span>(8)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/7/7d/T--Munich--Model_Equation_3.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/7/7d/T--Munich--Model_Equation_3.png"><span>(9)</span></div> |
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/3/35/T--Munich--Model_Equation_4.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/3/35/T--Munich--Model_Equation_4.png"><span>(10)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/8/81/T--Munich--Model_Equation_5.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/8/81/T--Munich--Model_Equation_5.png"><span>(11)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/6/6d/T--Munich--Model_Equation_6.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/6/6d/T--Munich--Model_Equation_6.png"><span>(12)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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<p> | <p> | ||
Equation 7 + 8 | Equation 7 + 8 | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/b/b7/T--Munich--Model_Equation_7.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/b/b7/T--Munich--Model_Equation_7.png"><span>(13)</span></div> |
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/e/e5/T--Munich--Model_Equation_8.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/e/e5/T--Munich--Model_Equation_8.png"><span>(14)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img style="height: 40px;" src="https://static.igem.org/mediawiki/2017/4/4e/T--Munich--Model_Equation_10.png"><span>( | + | <div class="equationDiv"><img style="height: 40px;" src="https://static.igem.org/mediawiki/2017/4/4e/T--Munich--Model_Equation_10.png"><span>(15)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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</p> | </p> | ||
<p> | <p> | ||
− | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/5/5d/T--Munich--Model_Equation_11.png"><span>( | + | <div class="equationDiv"><img src="https://static.igem.org/mediawiki/2017/5/5d/T--Munich--Model_Equation_11.png"><span>(16)</span></div> |
</p> | </p> | ||
<p> | <p> | ||
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<li id=“ref_10”>Licciardello, J. J., & Nickerson, J. T. R. (1963). “Some Observations on Bacterial Thermal Death Time Curves.” Applied Microbiology, 11(6), 476–480. | <li id=“ref_10”>Licciardello, J. J., & Nickerson, J. T. R. (1963). “Some Observations on Bacterial Thermal Death Time Curves.” Applied Microbiology, 11(6), 476–480. | ||
<li id=“ref_11”>Deindoerfer, F. H. (1957). “Calculation of Heat Sterilization Times for Fermentation Media.” Applied Microbiology, 5(4), 221–228. | <li id=“ref_11”>Deindoerfer, F. H. (1957). “Calculation of Heat Sterilization Times for Fermentation Media.” Applied Microbiology, 5(4), 221–228. | ||
+ | |||
+ | <li id="ref_12">M. Weitz, K. Jongmin, K. Kapsner, E. Winfree, E. Franco, and F.C. Simmel. | ||
+ | “Diversity in the Dynamical Behaviour of a Compartmentalized Programmable Biochemical Oscillator.” | ||
+ | (2014) <i>Nature Chemistry</i> 6(4): 295–302. | ||
+ | |||
+ | <li id="ref_13">V. Mekler1, L. Minakhin, E. Semenova, K. Kuznedelov and K. Severinov | ||
+ | "Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA" | ||
+ | <i>Nucleic Acids Research</i>, Vol. 44(6): 2837–2845 | ||
</ol> | </ol> |
Latest revision as of 03:53, 2 November 2017
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