Cas9 & Cpf1 secretion
and activity
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+ | <div style="position: absolute;top: 0;right: -250px;width: 200px;text-align: center;border: 1px solid gold;padding: 10px;border-radius: 10px;box-sizing: border-box;background: #ffedb8;"> | ||
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+ | ">Awards</div> | ||
+ | <img width="100" src="https://static.igem.org/mediawiki/2017/thumb/a/a2/UU_gold_medal.png/240px-UU_gold_medal.png"><br><div style="font-size: 15px;color: #c48b00; border-bottom: 1px solid #ffd700; padding-bottom: 15px; margin-top: 5px;">Gold medal</div> | ||
+ | <div style="margin-top: 15px; margin-bottom: 10px; font-size: 15px;color: #c48b00;"><b>Nominated</b><br />Best integrated human practices</div> | ||
+ | </div> | ||
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<div class="page-heading">The OUTCASST two-component system</div> | <div class="page-heading">The OUTCASST two-component system</div> | ||
− | This year | + | This year is the debut year for the Utrecht University iGEM team. Our team has developed an easy to use and cheap DNA detection kit for disease diagnosis in areas of the world where advanced diagnostic technologies are not available. We call our system ‘OUTCASST’, which stands for ‘Out-of-cell Crispr-Activated Sequence-specific Signal Transducer’. |
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<h2 class="subhead" id="subhead-2">The problem</h2> | <h2 class="subhead" id="subhead-2">The problem</h2> | ||
Disease diagnosis is of great importance for healthcare. | Disease diagnosis is of great importance for healthcare. | ||
− | In developing countries, diagnoses are often based on limited information, even though accurate disease determination based on pathogen specific DNA is possible through sequencing technologies. These technologies, however, require specialised equipment and expertise that simply is not available | + | In developing countries, diagnoses are often based on limited information, even though accurate disease determination based on pathogen specific DNA is possible through sequencing technologies. These technologies, however, require specialised equipment and expertise that simply is not available in developing parts of the world. |
− | + | The OUTCASST two-component system and detection kit was designed to alleviate this problem. | |
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− | + | First, guide RNA (gRNA) needs to be added, which is complementary to the DNA sequence you want to detect. | |
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− | + | dCas9 and dCpf1 will bind their corresponding gRNA. | |
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− | + | DNA from the sample that matches the gRNA will first bind to one of the proteins. | |
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− | + | Once the DNA fragment binds the other protein, the system will co-localize. This allows the protease to release the transcription factor from the complex, resulting in an intracellular signal. | |
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<h2 class="subhead" id="subhead-3">The system</h2> | <h2 class="subhead" id="subhead-3">The system</h2> | ||
− | The OUTCASST two-component system consists of two | + | The OUTCASST two-component system consists of two synthetic receptors that span the membrane. |
One of the proteins has a Cas9 protein attached as an extracellular domain, the other has a Cpf1 protein attached. | One of the proteins has a Cas9 protein attached as an extracellular domain, the other has a Cpf1 protein attached. | ||
Both proteins can be given a guide RNA that makes them bind to a specific, user-chosen, complementary sequence. | Both proteins can be given a guide RNA that makes them bind to a specific, user-chosen, complementary sequence. | ||
− | When both proteins bind a single DNA fragment from a sample, possibly containing pathogen DNA, they co-localize, so that a protease releases a transcription factor which then induces an intracellular reporter mechanism | + | When both proteins bind a single DNA fragment from a sample, possibly containing pathogen DNA, they co-localize, so that a protease releases a transcription factor which then induces an intracellular reporter mechanism such as a luminescent or fluorescent signal. |
+ | <br><br> | ||
+ | A final product would include the use of so-called anhydrobiotic insect <i>Polypedilum vanderplanki</i> cells, which can be air-dried, allowing them to be stored for prolonged periods of time at room temperature. The OUTCASST system is cheap to produce, store and ship, and requires nothing more then a simple microscope as a readout. | ||
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<script type="text/javascript" language="JavaScript"> | <script type="text/javascript" language="JavaScript"> | ||
function tut_goto(step) | function tut_goto(step) | ||
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{ | { | ||
jQuery("#link-" + i).removeClass("selected"); | jQuery("#link-" + i).removeClass("selected"); | ||
+ | jQuery("#link-" + i).removeClass("pulsing"); | ||
jQuery("#popover-" + i).dxPopover("hide"); | jQuery("#popover-" + i).dxPopover("hide"); | ||
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var title; | var title; | ||
− | if(i == 1) | + | /*if(i == 1) |
title = "Guide RNA"; | title = "Guide RNA"; | ||
else if(i == 2) | else if(i == 2) | ||
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else if(i == 3) | else if(i == 3) | ||
title = "Signal transduction"; | title = "Signal transduction"; | ||
+ | else if(i == 4) | ||
+ | title = "Signal transduction";*/ | ||
+ | |||
+ | if(i == 1) | ||
+ | title = "Start"; | ||
+ | else if(i == 2) | ||
+ | title = "gRNA binding"; | ||
+ | else if(i == 3) | ||
+ | title = "DNA binding"; | ||
else if(i == 4) | else if(i == 4) | ||
title = "Signal transduction"; | title = "Signal transduction"; | ||
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<br><br> | <br><br> | ||
Additionally, together with team Wageningen_UR a final experiment was done to verify that the protein in the medium was indeed secreted instead of due to involuntary cell lysis (see <a onclick="return change_page('collaborations', 1)" href="collaborations">Collaborations</a>). | Additionally, together with team Wageningen_UR a final experiment was done to verify that the protein in the medium was indeed secreted instead of due to involuntary cell lysis (see <a onclick="return change_page('collaborations', 1)" href="collaborations">Collaborations</a>). | ||
− | This experiment was done in duplo, by members from both team Wageningen_UR and team Utrecht, individually, to provide independent verification of the result. This final experiment was done according to a collaboration protocol that was shared with the Wageningen_UR team | + | This experiment was done in duplo, by members from both team Wageningen_UR and team Utrecht, individually, to provide independent verification of the result. This final experiment was done according to a collaboration protocol that was shared with the Wageningen_UR team <a target=_BLANK href="https://static.igem.org/mediawiki/2017/4/40/UuProtocolCollaborationWageningen.pdf" class="pdf pdf-inline"></a>. |
<br><br> | <br><br> | ||
<b>Endonuclease activity assay</b> | <b>Endonuclease activity assay</b> | ||
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<b>Endonuclease activity assay</b><br> | <b>Endonuclease activity assay</b><br> | ||
− | The first endonuclease activity assay was used to determine the optimal concentration of gRNA for the assay with the secreted proteins. The concentrations used were all functional for the Cpf1 protein (Figure 5). Cas9 however, showed no cleavage of the targeted DNA at all. This may be due to low quality gRNA. In the second assay, which contained sCas9 and sCpf1 purified from medium, there was no cleavage visible at all, even for the controls with Cas9 and Cpf1, as shown in figure | + | The first endonuclease activity assay was used to determine the optimal concentration of gRNA for the assay with the secreted proteins. The concentrations used were all functional for the Cpf1 protein (Figure 5, left pane). Cas9 however, showed no cleavage of the targeted DNA at all. This may be due to low quality gRNA. In the second assay, which contained sCas9 and sCpf1 purified from medium, there was no cleavage visible at all, even for the controls with Cas9 and Cpf1, as shown in figure 5 (right pane). |
− | <center><img style="margin-top: | + | <center><img style="margin-top: 25px;" src="https://static.igem.org/mediawiki/2017/f/fc/UU_secretion_fig5.png"></center> |
<span class="text-figure"> | <span class="text-figure"> | ||
<b>Figure 5.</b> Left - DNA gel electrophoresis of a linearized 800 base pair length plasmid. Concentrations of Cas9 and Cpf1 used in the assay were 0,05 uM and 0,15 uM, respectively. Right - DNA gel electrophoresis of the linearized 800bp plasmid. 2,5 nM Cas9 or Cpf1 protein was used and 10 nM gRNA. | <b>Figure 5.</b> Left - DNA gel electrophoresis of a linearized 800 base pair length plasmid. Concentrations of Cas9 and Cpf1 used in the assay were 0,05 uM and 0,15 uM, respectively. Right - DNA gel electrophoresis of the linearized 800bp plasmid. 2,5 nM Cas9 or Cpf1 protein was used and 10 nM gRNA. | ||
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<h2 class="subhead" id="subhead-6">Supplementary</h2> | <h2 class="subhead" id="subhead-6">Supplementary</h2> | ||
− | Plasmid nanodrop results can be | + | Plasmid nanodrop results can be downloaded here: <a target=_BLANK href="https://static.igem.org/mediawiki/2017/b/b1/UU_-_Assembly_supplementary.pdf" class="pdf pdf-inline"></a>. |
</script> | </script> | ||
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<br><br> | <br><br> | ||
By comparing the two figures, we can now see that the probability of cleavage for the slow cleaver is much smaller than that of the fast cleaver in the timespan that the transient complex persists. Of course, the concentration of the substrate-mediated complex decreases over time, so the total cleavage decreases when it cleaves later. To investigate how much the transient and substrate-mediated complex contribute to signal development for both Protease Chain variants, we define: | By comparing the two figures, we can now see that the probability of cleavage for the slow cleaver is much smaller than that of the fast cleaver in the timespan that the transient complex persists. Of course, the concentration of the substrate-mediated complex decreases over time, so the total cleavage decreases when it cleaves later. To investigate how much the transient and substrate-mediated complex contribute to signal development for both Protease Chain variants, we define: | ||
− | < | + | |
− | + | <center><img height="75" src="https://static.igem.org/mediawiki/2017/b/b5/UuModelingEquation1.png"></center> | |
− | + | ||
− | Wherein S’ is the increase in signal, given by the probability of cleavage ( | + | Wherein S’ is the increase in signal, given by the probability of cleavage (p<sub>c</sub>) for the remaining uncleaved complex. The remaining uncleaved complex is given by the remaining complex fraction (C) and how likely it is that it has not already been cleaved (one minus the integral of p<sub>c</sub> from 0 until that timepoint). |
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− | When we solve this for each Protease Chain version and for both the transient and substrate-mediated complex, we end up with time-plots of the signal contribution of a single binding event. In these plots, we see that the resulting substrate-mediated complex signal for the slow cleaver (top) is about half as strong as the signal for the fast cleaver (bottom).In theory, this is not a problem since the signal can be amplified by the cells. | + | When we solve this for each Protease Chain version and for both the transient and substrate-mediated complex, we end up with time-plots of the signal contribution of a single binding event. In these plots, we see that the resulting substrate-mediated complex signal for the slow cleaver (top) is about half as strong as the signal for the fast cleaver (bottom). In theory, this is not a problem since the signal can be amplified by the cells. |
<br><br> | <br><br> | ||
For the fast cleaver, we see a much bigger issue. The signal contribution of the transient complex, i.e. the false positive, is only about ten-fold smaller than the signal contribution of the substrate-mediated complex. Considering that transient encounters will be a lot more frequent than substrate-binding events, the false positive signal can be multiplied many times, making it a lot stronger than the substrate-mediated signal can ever be. | For the fast cleaver, we see a much bigger issue. The signal contribution of the transient complex, i.e. the false positive, is only about ten-fold smaller than the signal contribution of the substrate-mediated complex. Considering that transient encounters will be a lot more frequent than substrate-binding events, the false positive signal can be multiplied many times, making it a lot stronger than the substrate-mediated signal can ever be. | ||
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This is not the case for the slower cleaver, where the transient complex signal contribution is much smaller. | This is not the case for the slower cleaver, where the transient complex signal contribution is much smaller. | ||
<br><br> | <br><br> | ||
− | Using the contributions of a single transient binding event and a single substrate-mediated binding event, we can calculate a proxy for precision by dividing the contribution of the true signal (substrate-mediated) by the contribution of the false signal (transient). For the slow rate, the contribution of a single substrate-mediated event is almost 48 000 times that of the transient occurrence. For the fast cleavage rate, this is only 17 times. | + | Using the contributions of a single transient binding event and a single substrate-mediated binding event, we can calculate a proxy for precision by dividing the contribution of the true signal (substrate-mediated) by the contribution of the false signal (transient). For the slow rate, the contribution of a single substrate-mediated event is almost 48,000 times that of the transient occurrence. For the fast cleavage rate, this is only 17 times. |
<br><br> | <br><br> | ||
If we assume that the transient interaction occurs 100 times more frequently than the substrate-binding event, a modest estimate, ‘true’ signal strength would only be 0.17 times that of the background for the fast cleaver whereas it would still be 480 times stronger than the background for the slow cleaving protease. | If we assume that the transient interaction occurs 100 times more frequently than the substrate-binding event, a modest estimate, ‘true’ signal strength would only be 0.17 times that of the background for the fast cleaver whereas it would still be 480 times stronger than the background for the slow cleaving protease. | ||
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<br><br> | <br><br> | ||
− | There are eight important parameters in these equations. The | + | There are eight important parameters in these equations. The k<sub>1</sub> and k<sub>2</sub> rates are the association and dissociation rates between DNA and the target chain. The k<sub>3</sub> and k<sub>4</sub> are the association and dissociation rates of the protease chain and DNA. k<sub>5</sub> gives the effective cleavage rate. pP and pT are the production rates of the protease and target chain and d gives the decay-rate for proteins in general. |
<br><br> | <br><br> | ||
− | The concentration of each state is given by the name of the state in square brackets, such that T stands for target chain, S stands for substrate (DNA), P stands for protease chain, F stands for effector molecule and | + | The concentration of each state is given by the name of the state in square brackets, such that T stands for target chain, S stands for substrate (DNA), P stands for protease chain, F stands for effector molecule and T<sub>c</sub> stands for cleaved target chain. Binding is indicated by ‘:’ between two components. |
<br><br> | <br><br> | ||
− | The first thing we did was to check whether this system of equations behaves the way we expect it to, on short timescales. At first, we were only interested in the system equilibria. At the time-scale of state transitions, protein production and decay per time-unit are negligible and so the values for d, | + | The first thing we did was to check whether this system of equations behaves the way we expect it to, on short timescales. At first, we were only interested in the system equilibria. At the time-scale of state transitions, protein production and decay per time-unit are negligible and so the values for d, p<sub>T</sub> and p<sub>P</sub> were initially set to zero. |
<br><br> | <br><br> | ||
− | Using the association and dissociation constants from Richardson et al. <i class="ref" data-id="3">3</i>, we set the parameters | + | Using the association and dissociation constants from Richardson et al. <i class="ref" data-id="3">3</i>, we set the parameters k<sub>3</sub> and k<sub>4</sub> to 4 * 10<sup>4</sup> and 5 * 10<sup>-5</sup> respectively. Fonfara et al. <i class="ref" data-id="4">4</i> found a range of Cpf1 affinities in the same order of magnitude as Richardson et al. and so we chose to perform several runs with a variety of Cpf1 parameters going from .9 to 1.1 times the rates of Cas9. All runs resulted in similar equilibria where all target chain would be processed and cleaved. |
<br><br> | <br><br> | ||
This result is expected from a well-mixed system but, considering the membrane-bound nature of our proteins, the dynamics might not be accurate. Diffusion in the membrane is limited and thus might limit the interactions of the molecules. We made an attempt to illustrate this, too, using a molecular dynamics model but this will be described later on. | This result is expected from a well-mixed system but, considering the membrane-bound nature of our proteins, the dynamics might not be accurate. Diffusion in the membrane is limited and thus might limit the interactions of the molecules. We made an attempt to illustrate this, too, using a molecular dynamics model but this will be described later on. | ||
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From these relations, it is already clear that there can only be a stable signal potential, i.e. a stable fraction of the target chains remains uncleaved, when the production of target chain outweighs the decay and cleavage of it, whilst the acquisition of cleaved target chain is balanced by its decay, such that the following conditions apply: | From these relations, it is already clear that there can only be a stable signal potential, i.e. a stable fraction of the target chains remains uncleaved, when the production of target chain outweighs the decay and cleavage of it, whilst the acquisition of cleaved target chain is balanced by its decay, such that the following conditions apply: | ||
− | < | + | <center><img height="50" src="https://static.igem.org/mediawiki/2017/9/91/UuModelingEquation2.png"></center> |
− | + | <center><img height="45" src="https://static.igem.org/mediawiki/2017/e/e0/UuModelingEquation3.png"></center> | |
− | < | + | |
− | + | ||
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Which together forms: | Which together forms: | ||
− | <br>< | + | <br> |
− | + | <center><img height="45" src="https://static.igem.org/mediawiki/2017/9/90/UuModelingEquation4.png"></center> | |
− | < | + | <br> |
However, the concentrations of the intermediary complexes is dependent on the concentration and hence production of protease chain P. We know that, at equilibrium concentrations, the following must hold: | However, the concentrations of the intermediary complexes is dependent on the concentration and hence production of protease chain P. We know that, at equilibrium concentrations, the following must hold: | ||
<br><br> | <br><br> | ||
− | + | <center><img height="45" src="https://static.igem.org/mediawiki/2017/4/4b/UuModelingEquation5.png"></center> | |
− | < | + | <br> |
By combining these equations, we can get an expression for T depending on the production, decay and complex concentrations: | By combining these equations, we can get an expression for T depending on the production, decay and complex concentrations: | ||
<br><br> | <br><br> | ||
− | + | <center><img height="50" src="https://static.igem.org/mediawiki/2017/b/b9/UuModelingEquation6.png"></center> | |
− | < | + | <br> |
By assuming that the DNA binding dynamics of the system occur at much faster timescales than protein production and decay, we can assume that the substrate binding of the protease and target chains is at steady state, yielding the following expressions: | By assuming that the DNA binding dynamics of the system occur at much faster timescales than protein production and decay, we can assume that the substrate binding of the protease and target chains is at steady state, yielding the following expressions: | ||
<br><br> | <br><br> | ||
− | + | <center><img height="75" src="https://static.igem.org/mediawiki/2017/0/0d/UuModelingEquation7.png"></center> | |
+ | <center><img height="75" src="https://static.igem.org/mediawiki/2017/1/19/UuModelingEquation8.png"></center> | ||
+ | <center><img height="75" src="https://static.igem.org/mediawiki/2017/e/ee/UuModelingEquation9.png"></center> | ||
+ | <br> | ||
+ | We could then substitute these three concentrations for their expressions in the expression of the target chain concentration. Making further quasi steady state assumptions on the formation of the pre-cleavage and post-cleavage complexes reduces the expression by two more dependencies. This was done in mathematica notebook, found <a target=_BLANK href="https://static.igem.org/mediawiki/2017/2/21/UuModelingQSSAWorkouts.txt" class="url_external">here</a>. | ||
<br><br> | <br><br> | ||
− | + | ||
+ | The resulting expression shows that the concentration of target chain depends on: 1) The concentrations of its production relative to the production of the protease chain. 2) The concentration of protease chain. 3) The concentration of substrate. 4) How much cleaved target chain is available to trap said substrate. | ||
<br><br> | <br><br> | ||
− | + | The fraction of the total target chain that is cleaved is a saturation function that depends on substrate and protease chain concentrations with respect to how quickly the function's saturation point is attained. We can minimize the cleaved target chain fraction, and the occurrence of substrate trapping with it, by simply having a target chain amount that is much larger than that of the substrate. | |
<br><br> | <br><br> | ||
− | + | In short, the more substrate there is available per target chain, the less signal per substrate molecule we can get as ineffectual target chain concentration increases. | |
<br><br> | <br><br> | ||
− | + | The equations suggest that there is a theoretical optimum for the production rates of both chains, relative to the substrate concentration in the system. Due to time constraints, the expression for this optimum could not be given. The methods given in the mathematica script provided here should be able to reach this solution, given enough time. The meaning of such an optimum, however, is questionable. As the substrate concentrations in our toolkit may differ greatly depending on severity of infection and chance, optimization through growth-rates would need to be different per sample. In conclusion, the only effective optimization of protein productions is to make sure that the protein concentrations greatly exceed the sample concentration of DNA sequence we wish to detect. | |
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<script id="page-stakeholders" type="text/template"> | <script id="page-stakeholders" type="text/template"> | ||
− | As OUTCASST is a system with a very broad application, we wanted to find the most suitable end users and focus our design on their needs and restrictions. To achieve this goal, we have interviewed multiple professionals from different backgrounds. In these conversations, we tried to discover strengths and weaknesses of our system and design with regard to existing techniques and whether the OUTCASST system would be useful in the professions of the interviewed individuals. If not, we tried to figure out in what direction it might prove more useful or what aspects needed to be improved. In addition, we also discussed safety issues and technical aspects of our toolkit design. | + | As OUTCASST is a system with a very broad application, we wanted to find the most suitable end users and focus our design on their needs and restrictions. To achieve this goal, we have interviewed multiple professionals from different backgrounds. In these conversations, we tried to discover strengths and weaknesses of our system and design with regard to existing techniques and whether the OUTCASST system would be useful in the professions of the interviewed individuals. If not, we tried to figure out in what direction it might prove more useful or what aspects needed to be improved. In addition, we also discussed safety issues and technical aspects of our toolkit design, of which the outcomes were compiled on their own respective pages: <a onclick="return change_page('safety', 1)" href="safety">Safety</a> and <a onclick="return change_page('product-design', 1)" href="product-design">Design & Integration</a>. |
<br><br> | <br><br> | ||
In the short video below, we show a summary of the journey we made to get to our end users. Besides this summary, more elaborate descriptions of each interview can be found below. | In the short video below, we show a summary of the journey we made to get to our end users. Besides this summary, more elaborate descriptions of each interview can be found below. | ||
<br><br> | <br><br> | ||
− | <video style="width: 100%;" poster="" controls> | + | |
− | <source src=" | + | <video onclick="this.paused?this.play():this.pause();" style="width: 100%; cursor: pointer;" poster="https://static.igem.org/mediawiki/2017/8/8d/UU-stakeholders-video-poster.png" controls> |
− | + | <source src="https://static.igem.org/mediawiki/2017/6/6f/UuHPEndUsersVid.mp4" type='video/mp4'/> | |
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</video> | </video> | ||
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<div class="aspect aspect--32x9"> | <div class="aspect aspect--32x9"> | ||
<div class="aspect__inner"><img src="https://static.igem.org/mediawiki/2017/f/fe/Marit_de_Wit.jpg"></div> | <div class="aspect__inner"><img src="https://static.igem.org/mediawiki/2017/f/fe/Marit_de_Wit.jpg"></div> | ||
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<div class="aspect aspect--9x80"> | <div class="aspect aspect--9x80"> | ||
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<div class="aspect aspect--32x9"> | <div class="aspect aspect--32x9"> | ||
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− | <figure class="js-item column col-span shuffle-item shuffle-item--visible" style="position: absolute; top: 0px; left: 0px; visibility: visible; will-change: transform; opacity: 1; transform: translate(0px, 389px) scale(1); transition-duration: 250ms; transition-timing-function: cubic-bezier(0.4, 0, 0.2, 1); transition-property: transform, opacity;"> | + | <figure class="js-item column col-span shuffle-item shuffle-item--visible" onclick="jQuery('html, body').animate({scrollTop: jQuery('#item-genomediagnostics').offset().top - 150}, 500);" style="cursor: pointer; position: absolute; top: 0px; left: 0px; visibility: visible; will-change: transform; opacity: 1; transform: translate(0px, 389px) scale(1); transition-duration: 250ms; transition-timing-function: cubic-bezier(0.4, 0, 0.2, 1); transition-property: transform, opacity;"> |
<div class="aspect aspect--32x9"> | <div class="aspect aspect--32x9"> | ||
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<div class="aspect aspect--9x80"> | <div class="aspect aspect--9x80"> | ||
<div class="aspect__inner"><img src="https://static.igem.org/mediawiki/2017/5/59/Hugo_Snippert_%282%29.jpg"></div> | <div class="aspect__inner"><img src="https://static.igem.org/mediawiki/2017/5/59/Hugo_Snippert_%282%29.jpg"></div> | ||
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− | <figure class="js-item column shuffle-item shuffle-item--visible" style="position: absolute; top: 0px; left: 0px; visibility: visible; will-change: transform; opacity: 1; transform: translate(675px, 260px) scale(1); transition-duration: 250ms; transition-timing-function: cubic-bezier(0.4, 0, 0.2, 1); transition-property: transform, opacity;"> | + | <figure class="js-item column shuffle-item shuffle-item--visible" onclick="jQuery('html, body').animate({scrollTop: jQuery('#item-nfi').offset().top - 150}, 500);" style="cursor: pointer; position: absolute; top: 0px; left: 0px; visibility: visible; will-change: transform; opacity: 1; transform: translate(675px, 260px) scale(1); transition-duration: 250ms; transition-timing-function: cubic-bezier(0.4, 0, 0.2, 1); transition-property: transform, opacity;"> |
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+ | </div> | ||
+ | </figure> | ||
+ | <figure class="js-item column shuffle-item shuffle-item--visible" style="position: absolute; top: 0px; left: 0px; visibility: visible; will-change: transform; opacity: 1; transform: translate(450px, 389px) scale(1); transition-duration: 250ms; transition-timing-function: cubic-bezier(0.4, 0, 0.2, 1); transition-property: transform, opacity;"> | ||
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<div class="page-heading">Integrated human practices: design of OUTCASST</div> | <div class="page-heading">Integrated human practices: design of OUTCASST</div> | ||
Altogether, we gathered information from many different fields and perspectives. All of these views helped us to optimize our design to use OUTCASST as a tool for diagnosing Chagas disease. Since we want our tool to be easy to use in the field and rural areas, there are different aspects that should be taken into account. Robustness and resistance of the toolkit to temperature fluctuations and humidity are chief among these. In addition, it is important not to rely on material and storage containers such as fridges or freezers (Marit de Wit, Doctors without borders). | Altogether, we gathered information from many different fields and perspectives. All of these views helped us to optimize our design to use OUTCASST as a tool for diagnosing Chagas disease. Since we want our tool to be easy to use in the field and rural areas, there are different aspects that should be taken into account. Robustness and resistance of the toolkit to temperature fluctuations and humidity are chief among these. In addition, it is important not to rely on material and storage containers such as fridges or freezers (Marit de Wit, Doctors without borders). | ||
<br><br> | <br><br> | ||
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+ | |||
+ | <div style="clear: both;"></div> | ||
+ | |||
+ | <div id="popover-1" style="display: none;"> | ||
+ | The OUTCASST toolkit consists of dried cells in a closed off compartment, two medium compartments (green) and a lysis and reset buffer compartment (purple). | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-2" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-2" style="display: none;"> | ||
+ | First, the seal on one of the medium compartments is broken so medium goes onto the cells. The cells will now be rejuvenated and allowed to acclimatize in 12 hours. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-3" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-3" style="display: none;"> | ||
+ | After 12 hours, a patient's sample can be added to the test kit. The sample will enter the filtering compartment, which contains chemicals that bind all sorts of unwanted chemicals. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-4" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-4" style="display: none;"> | ||
+ | The seal on the lysis buffer compartment is broken. The lysis buffer causes any intact cells to burst, releasing their DNA and internal contents. Unwanted cell debris is bound by the filter molecules, coated to the inside of the filtering compartment. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-5" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-5" style="display: none;"> | ||
+ | The seal on the reset buffer compartment is broken. Reset buffer, sample and lysis buffer mingle, neutralizing each other and resulting in an isotonic mixture that will not harm the sensor cells. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-6" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-6" style="display: none;"> | ||
+ | The second medium pocket is used to resuspend the gRNA. We kept the gRNA dry until now to prevent degradation of this sensitive chemical. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-7" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-7" style="display: none;"> | ||
+ | The dissolved gRNA is released onto the sensor cells. The protein chains on the surface of these cells can now bind with the gRNA, making the sensor cells specific for DNA that is complementary to the gRNA they were provided with. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-8" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
+ | </div> | ||
+ | |||
+ | <div id="popover-8" style="display: none;"> | ||
+ | The seal to the waste compartment is broken. As this compartment was under a slight vacuum, a part of the medium is sucked away from the cells, making room for the sample. | ||
+ | <br> | ||
+ | <br> | ||
+ | <a href="javascript: void(0);" class="btn blue" id="goto-9" style="margin: 0; padding: 10px 25px; font-size: 18px;">Next</a> | ||
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+ | |||
+ | <div id="popover-9" style="display: none;"> | ||
+ | The seal to the waste compartment is closed again and so are the seals to the medium pockets. The lysed and filtered sample, containing the patient's DNA, is now brought to the cells. If the right DNA sequence is present, the cells will detect it and give an output signal. | ||
+ | </div> | ||
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The tool should be cheap to produce and easy to use. To achieve this goal, the tool needs to have a closed box design wherein only the blood sample has to be added and a simple protocol can be followed to perform the test (Jet Bliek and Ruud van den Bogaard, Academical Medical Center Amsterdam: clinical genetics). Disposal of the system also needs to be considered (collaboration RIVM National Insitute for Public Health and the Environment). | The tool should be cheap to produce and easy to use. To achieve this goal, the tool needs to have a closed box design wherein only the blood sample has to be added and a simple protocol can be followed to perform the test (Jet Bliek and Ruud van den Bogaard, Academical Medical Center Amsterdam: clinical genetics). Disposal of the system also needs to be considered (collaboration RIVM National Insitute for Public Health and the Environment). | ||
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The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them. | The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them. | ||
<br><br> | <br><br> | ||
− | As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration | + | As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration <i class="ref" data-id="1">1</i>. This way the shelf life of our tool can also be prolonged. To prevent the risk of our GMO getting out in the environment, several mechanisms and kill-switches will be incorporated in the cells, so they can only survive in our closed box system, in their resurgent state. |
This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance. | This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance. | ||
<br><br> | <br><br> | ||
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We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. | We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. | ||
We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the <i>T. cruzi</i> parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist). | We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the <i>T. cruzi</i> parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist). | ||
+ | |||
+ | <br><br> | ||
+ | <h2 class="subhead" id="subhead-4">References</h2> | ||
+ | |||
+ | <ol class="references"> | ||
+ | <li data-title="Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term…" data-author="Watanabe K, Imanishi S, Akiduki G, Cornette R, Okuda T." data-link="https://doi.org/10.1016/j.cryobiol.2016.05.006" /> Watanabe K, Imanishi S, Akiduki G, Cornette R, Okuda T. Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration. Cryobiology. 2016 Aug 31;73(1):93-8. <a target=_BLANK href="https://doi.org/10.1016/j.cryobiol.2016.05.006" class="url_external"></a> | ||
+ | </ol> | ||
+ | |||
+ | <span id="tooltip-1"></span> | ||
--></script> | --></script> | ||
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<script id="page-outreach" type="text/template"> | <script id="page-outreach" type="text/template"> | ||
<div class="page-heading">Outreach</div> | <div class="page-heading">Outreach</div> | ||
− | Science | + | Science impacts the world in many ways. With our project, we are not only aiming to make a difference by creating a diagnostic tool, but also to reach out to the public to create awareness and make science accessible for everyone. We collaborated with ‘de Kennis van Nu’, a well-known national TV program and internet platform that brings different scientific themes to the general public in an understandable way. They aim to make science accessible to everyone, old and young, and encourage everyone to be curious and bring out the scientist in themselves! |
On their platform, we explain the formation of Utrecht’s very first team, our design and how we are trying to solve healthcare problems. | On their platform, we explain the formation of Utrecht’s very first team, our design and how we are trying to solve healthcare problems. | ||
− | Through our whole iGEM experience, they follow us from lab bench to Boston. | + | Through our whole iGEM experience, they follow us from lab bench to Boston. Their special about our team can be found <a target=_BLANK href="https://dekennisvannu.nl/site/special/iGEM-2017-studenten-ontwerpen-nieuw-leven/111#!/" class="url_external">here</a>. |
<br><br> | <br><br> | ||
Below, you can find the short movies, articles and infographics that were so far made in cooperation with Kennis van Nu to reach out to the public. | Below, you can find the short movies, articles and infographics that were so far made in cooperation with Kennis van Nu to reach out to the public. | ||
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<h2 class="subhead" id="subhead-2">iGEM Utrecht: an introduction</h2> | <h2 class="subhead" id="subhead-2">iGEM Utrecht: an introduction</h2> | ||
− | <video poster="" controls> | + | <video poster="https://static.igem.org/mediawiki/2017/2/2e/UU-poster-video1.png" controls> |
− | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/b/bb/UuHPOutreach1.mp4" type='video/mp4'/> |
− | + | ||
− | + | ||
− | + | ||
<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
+ | <div class="description">What does iGEM mean and what’s so fun about it? What are the biggest challenges faced during the experiments. In this movie we explain it all.</div> | ||
</div> | </div> | ||
<div class="outreach-video right"> | <div class="outreach-video right"> | ||
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<h2 class="subhead" id="subhead-2">Meet the team</h2> | <h2 class="subhead" id="subhead-2">Meet the team</h2> | ||
− | <video poster="" controls> | + | <video poster="https://static.igem.org/mediawiki/2017/6/6a/UU-poster-video2.png" controls> |
− | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/6/68/UuHPOutreach2.mp4" type='video/mp4'/> |
− | + | ||
− | + | ||
− | + | ||
<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
+ | |||
+ | <div class="description">During the formation of the team, a lot of attention is paid to diversity of the members and their background. In this movie some of the members introduce themselves and explain why they participate in this competition.</div> | ||
</div> | </div> | ||
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<h2 class="subhead" id="subhead-2">The problem; the detection of infectious disease has to be improved</h2> | <h2 class="subhead" id="subhead-2">The problem; the detection of infectious disease has to be improved</h2> | ||
− | <video poster="" controls> | + | <video poster="https://static.igem.org/mediawiki/2017/6/6c/Uu-outreach-video3-poster.png" controls> |
− | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/b/bb/UuHPOutreach3.mp4" type='video/mp4'/> |
− | + | ||
− | + | ||
− | + | ||
<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
+ | |||
+ | <div class="description">For which problem do we find a solution in our research? The concept of the problem is explained in this draw-my-life video.</div> | ||
</div> | </div> | ||
<div class="outreach-video right"> | <div class="outreach-video right"> | ||
− | <h2 class="subhead" id="subhead-2"> | + | <h2 class="subhead" id="subhead-2">Labsafety for Dummies</h2> |
− | <video poster="" controls> | + | <video poster="https://static.igem.org/mediawiki/2017/5/5b/UU-poster-video4.png" controls> |
− | <source src=" | + | <source src="https://static.igem.org/mediawiki/2017/4/4c/UuHPOutreach4.mp4" type='video/mp4'/> |
− | + | ||
− | + | ||
− | + | ||
<p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | <p style="font-style:italic;color:red;border-style:solid;border-width:2px;border-color:red">Your browser either does not support HTML5 or cannot handle MediaWiki open video formats. Please consider upgrading your browser, installing the appropriate plugin or switching to a Firefox or Chrome install.</p> | ||
</video> | </video> | ||
+ | |||
+ | <div class="description">The lab can be a treacherous environment. One tiny mistake, and it could be your last.. We answer important questions about how to stay safe in the lab.</div> | ||
</div> | </div> | ||
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.outreach-video>video { width: 100%; } | .outreach-video>video { width: 100%; } | ||
.outreach-video>h2 { font-size: 17px; color: black; font-weight: bold; padding-bottom: 10px; border-bottom: 2px solid #f6f6f6; } | .outreach-video>h2 { font-size: 17px; color: black; font-weight: bold; padding-bottom: 10px; border-bottom: 2px solid #f6f6f6; } | ||
+ | |||
+ | .outreach-video .description { font-size: 12px; line-height: 20px; display: inline-block; } | ||
</style> | </style> | ||
</script> | </script> | ||
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<span class="team-duties"> | <span class="team-duties"> | ||
› Experimental - Secreted Cas9 and Cpf1<br> | › Experimental - Secreted Cas9 and Cpf1<br> | ||
− | › Human Practices - Application | + | › Human Practices - Application<br> |
+ | › Biobricks | ||
</span> | </span> | ||
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› Human Practices -<br> Application/Safety/Design<br> | › Human Practices -<br> Application/Safety/Design<br> | ||
› Experimental - Assembly/InterLab Study<br> | › Experimental - Assembly/InterLab Study<br> | ||
− | › Funding & Sponsoring | + | › Funding & Sponsoring<br> |
+ | › Treasurer | ||
</span> | </span> | ||
<div style="margin-top: 10px; font-weight: bold;">Bio</div> | <div style="margin-top: 10px; font-weight: bold;">Bio</div> | ||
− | <div class="team-bio"> | + | <div class="team-bio" style="height: 61px;"> |
Hi! I'm Lishi (21), a first year master student in Pharmacy at Utrecht University. After that, I want to become a hospital pharmacist. | Hi! I'm Lishi (21), a first year master student in Pharmacy at Utrecht University. After that, I want to become a hospital pharmacist. | ||
<div class="lb"></div> | <div class="lb"></div> | ||
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</div> | </div> | ||
<span style="display: block; font-size: 12px; padding: 0 20px;"> | <span style="display: block; font-size: 12px; padding: 0 20px;"> | ||
− | + | Professor of Regenerative Medicine<br> | |
− | + | ||
<div style="margin-top: 10px; font-weight: bold;">Bio</div> | <div style="margin-top: 10px; font-weight: bold;">Bio</div> | ||
− | <div class="team-bio" style="height: | + | <div class="team-bio" style="height: 239px;"> |
− | + | Hello, I’m Niels Geijsen, and supervising the very first iGEM team of Utrecht University has been an immensely fun and inspiring adventure. Scientific challenges require for our brightest minds with diverse perspectives and backgrounds to come together. This is the Utrecht iGEM team: they are a group of smart, creative and ambitious students who have contributed a wide range of knowledge backgrounds, life experiences and hobbies to the project. It was such a fantastic experience to see how each member brought a special talent ranging from artistic drawing skills and video animation to photography, coding and web design. Together, they have truly outdone themselves. | |
+ | <div class="lb"></div> | ||
+ | As a professor in regenerative medicine at the Hubrecht Institute and Utrecht University, my research team works work at the boundaries of stem cell biology and technology development with a particular focus on (genetic) muscle disease. I spent 10 years in Boston, as post-doctoral fellow at the Whitehead Institute and faculty at Massachusetts General Hospital, Harvard Medical School and the Harvard Stem Cell Institute, and I'm particularly looking forward to joining the team in Boston for the Giant Jamboree. | ||
+ | <div class="lb"></div> | ||
+ | Aside from science, I experiment in the kitchen, have a passion for food and wine, and enjoy outdoor activities such as sailing, hiking and skiing. My three active young boys are a daily joy and challenge. <br> | ||
<div class="team-bio-grad"></div> | <div class="team-bio-grad"></div> | ||
</div> | </div> | ||
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</span> | </span> | ||
</div> | </div> | ||
− | <div class="back"></div></div> | + | <div class="back" style="line-height: 15px; font-size: 11px;"></div></div> |
</div> | </div> | ||
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<div class="team-bio" style="height: 215px;"> | <div class="team-bio" style="height: 215px;"> | ||
Hi all, I am Roos Masereeuw, and since June 2015 I am full professor of Experimental Pharmacology at Utrecht Institute for Pharmaceutical Sciences in The Netherlands. I did a master’s in Biopharmaceutical Sciences at Leiden University, and my PhD in Pharmacology and Toxicology at Radboud University in Nijmegen (January 1997). | Hi all, I am Roos Masereeuw, and since June 2015 I am full professor of Experimental Pharmacology at Utrecht Institute for Pharmaceutical Sciences in The Netherlands. I did a master’s in Biopharmaceutical Sciences at Leiden University, and my PhD in Pharmacology and Toxicology at Radboud University in Nijmegen (January 1997). | ||
− | < | + | <div class="lb"></div> |
Part of my PhD research and a postdoc period I did at National Institute for Environmental Sciences (NIEHS/NIH) in Research Triangle Park, North Carolina of USA. Between 1998 and 2015, I was first assistent and then associate professor at Radboud University Medical Centre in Nijmegen, after which I moved to Utrecht University. | Part of my PhD research and a postdoc period I did at National Institute for Environmental Sciences (NIEHS/NIH) in Research Triangle Park, North Carolina of USA. Between 1998 and 2015, I was first assistent and then associate professor at Radboud University Medical Centre in Nijmegen, after which I moved to Utrecht University. | ||
− | < | + | <div class="lb"></div> |
My research is focused on understanding the pathways that can be pharmacologically triggered to enhance repair and regeneration processes after organ injury. An example is the development of a bioartificial kidney device to partly replace kidney function. Next to science, I mostly enjoy outdoor sports when I can. This includes mountain hiking, mountain biking and running, but I also love watching Netflix series with my family, and visiting concerts and festivals. | My research is focused on understanding the pathways that can be pharmacologically triggered to enhance repair and regeneration processes after organ injury. An example is the development of a bioartificial kidney device to partly replace kidney function. Next to science, I mostly enjoy outdoor sports when I can. This includes mountain hiking, mountain biking and running, but I also love watching Netflix series with my family, and visiting concerts and festivals. | ||
<div class="team-bio-grad"></div> | <div class="team-bio-grad"></div> | ||
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<div class="team-bio" style="height: 201px;"> | <div class="team-bio" style="height: 201px;"> | ||
Hello, I’m Guido van den Ackerveken and it is my privilege to be one of the supervisors of the energetic and bright team of Utrecht University. I am trained as a molecular biologist in the field of plant- and microbiology, and have always worked of plant-pathogen interactions. I have a research team of about 10 people and in our research we reveal molecular mechanisms in the dialogue between plants and microorganisms, which we then try to translate to improve the resistance of food crops to infectious diseases. It is real fun to work with the iGEM bunch and to support the starting researchers in their ambitious programme. | Hello, I’m Guido van den Ackerveken and it is my privilege to be one of the supervisors of the energetic and bright team of Utrecht University. I am trained as a molecular biologist in the field of plant- and microbiology, and have always worked of plant-pathogen interactions. I have a research team of about 10 people and in our research we reveal molecular mechanisms in the dialogue between plants and microorganisms, which we then try to translate to improve the resistance of food crops to infectious diseases. It is real fun to work with the iGEM bunch and to support the starting researchers in their ambitious programme. | ||
− | < | + | <div class="lb"></div> |
Outside of my busy work I try to find time for playing squash and running (I ran my first marathon this year), but also of course to enjoy being with my family. Never a dull moment :-) | Outside of my busy work I try to find time for playing squash and running (I ran my first marathon this year), but also of course to enjoy being with my family. Never a dull moment :-) | ||
<div class="team-bio-grad"></div> | <div class="team-bio-grad"></div> | ||
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<div class="team-bio" style="height: 215px;"> | <div class="team-bio" style="height: 215px;"> | ||
I am a microbiologist and work at Utrecht University. My research interest focusses on two topics. One is to unravel the mechanisms involved in secretion of proteins and the role of secreted proteins in bacteria. This knowledge is important to understand how bacteria interact with their environment and can be used to optimize large scale production of proteins and vaccine development. | I am a microbiologist and work at Utrecht University. My research interest focusses on two topics. One is to unravel the mechanisms involved in secretion of proteins and the role of secreted proteins in bacteria. This knowledge is important to understand how bacteria interact with their environment and can be used to optimize large scale production of proteins and vaccine development. | ||
− | < | + | <div class="lb"></div> |
My second topic of interest is teaching, and especially how to stimulate students to become independent and critical researchers. Therefore, assisting the first Utrecht iGEM team has been a great experience. In my spare time, I like to eat, watch movies and spend time with my family, which includes two daughters and a lazy cat. | My second topic of interest is teaching, and especially how to stimulate students to become independent and critical researchers. Therefore, assisting the first Utrecht iGEM team has been a great experience. In my spare time, I like to eat, watch movies and spend time with my family, which includes two daughters and a lazy cat. | ||
<div class="team-bio-grad"></div> | <div class="team-bio-grad"></div> | ||
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<div class="team-bio" style="height: 201px;"> | <div class="team-bio" style="height: 201px;"> | ||
Hello, my name is Clara (23) and I’m a first year Cancer, Stem Cells and Developmental biology Master student in Utrecht. I finished a Bachelor’s degree in Genetics in Barcelona last year and came to the Netherlands with a fellowship for post-graduate internships abroad, which allowed me to join Niels Geijsen’s lab for six months. | Hello, my name is Clara (23) and I’m a first year Cancer, Stem Cells and Developmental biology Master student in Utrecht. I finished a Bachelor’s degree in Genetics in Barcelona last year and came to the Netherlands with a fellowship for post-graduate internships abroad, which allowed me to join Niels Geijsen’s lab for six months. | ||
− | < | + | <div class="lb"></div> |
I’m really happy to have been asked to write a piece as part of the team, even if I haven’t been there for the whole process. I joined the team during the summer period to help with some lab work that was being carried out in my former lab. It has been a great experience to help supervising the Utrecht iGEM team and its amazing people, full of joy and stress I have to admit. Now the deadline is getting closer and we are all preparing for the big final! Thank you, guys, for this opportunity! | I’m really happy to have been asked to write a piece as part of the team, even if I haven’t been there for the whole process. I joined the team during the summer period to help with some lab work that was being carried out in my former lab. It has been a great experience to help supervising the Utrecht iGEM team and its amazing people, full of joy and stress I have to admit. Now the deadline is getting closer and we are all preparing for the big final! Thank you, guys, for this opportunity! | ||
<div class="team-bio-grad"></div> | <div class="team-bio-grad"></div> | ||
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<div class="slider_for" style="float: left; width: 400px;"> | <div class="slider_for" style="float: left; width: 400px;"> | ||
<div><b>Utrecht University is a Dutch public university with over 50.000 students.</b> As our home university, they were our first sponsor. They have helped us generously with great financial support, meeting space, PR-facilities and the opportunity to use several labspaces on campus.</div> | <div><b>Utrecht University is a Dutch public university with over 50.000 students.</b> As our home university, they were our first sponsor. They have helped us generously with great financial support, meeting space, PR-facilities and the opportunity to use several labspaces on campus.</div> | ||
+ | <div><b>The Jong Alumni Netwerk (JAN) organizes activities and events to help support the careers of recent graduates by providing networking opportunities.</b> JAN agreed to financially support us to make this project possible.</div> | ||
+ | <div><b>The Hubrecht Institute is a leading research centre focusing on developmental biology and stem cell research.</b> Hubrecht institute offered us guidance and assistance with our project by facilitating a lab assistant for the work we had to do for our project.</div> | ||
<div><b>RIVM is the National Institute for Public Health and the Environment, which belongs to the Ministry of Health, Welfare and Sport of the Dutch government.</b> They offered a sponsorship to the Dutch iGEM teams based on an assignment titled ‘think before you do’, stimulating us to think about the societal implications that our project could have. We submitted a proposal and the RIVM offered us a €1500 grant to further our project.</div> | <div><b>RIVM is the National Institute for Public Health and the Environment, which belongs to the Ministry of Health, Welfare and Sport of the Dutch government.</b> They offered a sponsorship to the Dutch iGEM teams based on an assignment titled ‘think before you do’, stimulating us to think about the societal implications that our project could have. We submitted a proposal and the RIVM offered us a €1500 grant to further our project.</div> | ||
<div><b>DSM is a science-based company focusing on health, nutrition and materials to drive sustainable innovation.</b> DSM is active in many different markets, including medicine, energy and food, so developments in synthetic biology are of major interest to them. For our project, they sponsored us with €1000.</div> | <div><b>DSM is a science-based company focusing on health, nutrition and materials to drive sustainable innovation.</b> DSM is active in many different markets, including medicine, energy and food, so developments in synthetic biology are of major interest to them. For our project, they sponsored us with €1000.</div> | ||
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<div class="sponsors_slider" style="width: 100%;"> | <div class="sponsors_slider" style="width: 100%;"> | ||
<div style="width: 150px;"><img src="https://static.igem.org/mediawiki/2017/e/e0/Uu17logo.png"></div> | <div style="width: 150px;"><img src="https://static.igem.org/mediawiki/2017/e/e0/Uu17logo.png"></div> | ||
+ | <div style="width: 250px;"><img src="https://static.igem.org/mediawiki/2017/8/85/Uu-jongealumninetwerk.png"></div> | ||
+ | <div style="width: 200px;"><img src="https://static.igem.org/mediawiki/2017/2/2a/Uu-logo-hubrecht.png"></div> | ||
<div style="width: 300px;"><img src="https://static.igem.org/mediawiki/2017/e/e6/Uurivm.png"></div> | <div style="width: 300px;"><img src="https://static.igem.org/mediawiki/2017/e/e6/Uurivm.png"></div> | ||
<div style="width: 250px;"><img src="https://static.igem.org/mediawiki/2017/5/5b/Uudsm.jpeg"></div> | <div style="width: 250px;"><img src="https://static.igem.org/mediawiki/2017/5/5b/Uudsm.jpeg"></div> | ||
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Our team collaborated with the Wageningen team to obtain independent validations of both our and their biobricks. | Our team collaborated with the Wageningen team to obtain independent validations of both our and their biobricks. | ||
<br><br> | <br><br> | ||
+ | <div style="float: right; margin-left: 35px; margin-bottom: 10px;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/thumb/f/f5/Collab_WUR_UU.jpg/320px-Collab_WUR_UU.jpg"> | ||
+ | <br> | ||
+ | <span class="text-figure"> | ||
+ | Our collaboration with Wageningen has been great fun. | ||
+ | </span> | ||
+ | </div> | ||
Wageningen validated the secreted-Cas9 and secreted-Cpf1 biobricks for us (BB_numbers). | Wageningen validated the secreted-Cas9 and secreted-Cpf1 biobricks for us (BB_numbers). | ||
Members of the Wageningen team came to Utrecht to perform this validation together with members of the Utrecht team in our lab. | Members of the Wageningen team came to Utrecht to perform this validation together with members of the Utrecht team in our lab. | ||
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Medium of the cells was analysed on a western blot to compare extracellular protein expression. | Medium of the cells was analysed on a western blot to compare extracellular protein expression. | ||
This tells us whether Cas9 and Cpf1 are truly being secreted or if they just leak out of the cells. | This tells us whether Cas9 and Cpf1 are truly being secreted or if they just leak out of the cells. | ||
− | The results of this validation can be found on the <a onclick="return change_page(' | + | The results of this validation can be found on the <a onclick="return change_page('basic_part', 1)" href="basic_part">Parts Page</a> and the experimental page on <a onclick="return change_page('secretion', 1)" href="secretion">secreted Cas9 and Cpf1</a>. |
<br><br> | <br><br> | ||
In return, for the Wageningen team, we did a blind validation of some of their biobricks. | In return, for the Wageningen team, we did a blind validation of some of their biobricks. | ||
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We also performed a leucine zipper BiFC measurement, where we transformed split sfGFP and split Venus to E.coli K12 and also full sfGFP and full Venus as controls. | We also performed a leucine zipper BiFC measurement, where we transformed split sfGFP and split Venus to E.coli K12 and also full sfGFP and full Venus as controls. | ||
Cells were then lysed and fluorescence was measured in the cell lysate <a target=_BLANK href="https://2017.igem.org/Team:Wageningen_UR/Collaborations" class="url_external">(link)</a>. | Cells were then lysed and fluorescence was measured in the cell lysate <a target=_BLANK href="https://2017.igem.org/Team:Wageningen_UR/Collaborations" class="url_external">(link)</a>. | ||
− | |||
− | |||
− | |||
<br><br> | <br><br> | ||
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<br><br> | <br><br> | ||
− | < | + | |
+ | <center> | ||
+ | <img onclick="openmodal('rivm-event')" style="cursor: pointer;" src="https://static.igem.org/mediawiki/2017/thumb/f/f7/8997_Kennisparade_%281%29.jpg/240px-8997_Kennisparade_%281%29.jpg"> | ||
+ | <br> | ||
+ | <span class="text-figure"> | ||
+ | Attending a RIVM event. <a href="javascript:void(0)" onclick="openmodal('rivm-event')" style="font-size: 11px;">Click for full size.</a> | ||
+ | </span> | ||
+ | </center> | ||
<br><br> | <br><br> | ||
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<br><br> | <br><br> | ||
− | < | + | |
+ | <center> | ||
+ | <img width="100%" src="https://static.igem.org/mediawiki/2017/thumb/b/be/Collab_dusseldorf_postcards.jpg/800px-Collab_dusseldorf_postcards.jpg"> | ||
+ | </center> | ||
</script> | </script> | ||
<script id="page-achievements" type="text/template"> | <script id="page-achievements" type="text/template"> | ||
+ | <div style="position: absolute;top: 0;right: -250px;width: 200px;text-align: center;border: 1px solid gold;padding: 10px;border-radius: 10px;box-sizing: border-box;background: #ffedb8;"> | ||
+ | <div style=" | ||
+ | font-weight: bold; | ||
+ | font-size: 20px; | ||
+ | color: #c48b00; | ||
+ | border-bottom: 1px solid #ffd700; | ||
+ | padding-bottom: 15px; | ||
+ | margin-bottom: 15px; | ||
+ | ">Awards</div> | ||
+ | <img width="100" src="https://static.igem.org/mediawiki/2017/thumb/a/a2/UU_gold_medal.png/240px-UU_gold_medal.png"><br><div style="font-size: 15px;color: #c48b00; border-bottom: 1px solid #ffd700; padding-bottom: 15px; margin-top: 5px;">Gold medal</div> | ||
+ | <div style="margin-top: 15px; margin-bottom: 10px; font-size: 15px;color: #c48b00;"><b>Nominated</b><br />Best integrated human practices</div> | ||
+ | </div> | ||
<div class="page-heading">Achievements</div> | <div class="page-heading">Achievements</div> | ||
− | This page | + | This page gives an overview of the achievements of our team. These will be presented as the medal criteria we fulfilled to acquire the various medals. For several criteria we will provide a link to the page with more information on that item. |
<br><br> | <br><br> | ||
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<b>Deliverables</b><br> | <b>Deliverables</b><br> | ||
We have delivered all the required items on the iGEM deliverables page.<br> | We have delivered all the required items on the iGEM deliverables page.<br> | ||
− | <table border="0" cellspacing="0" cellpadding="0" width="600"> | + | <table class="uu-table" border="0" cellspacing="0" cellpadding="0" width="600" style="margin-top: 10px !important;"> |
<tr> | <tr> | ||
<td width="250"> | <td width="250"> | ||
<ul> | <ul> | ||
− | <li /><a href="">Team wiki</a> | + | <li /><a onclick="return change_page('home', 1)" href="https://2017.igem.org/Team:Utrecht/">Team wiki</a> |
− | <li /><a href="">Project attribution</a> | + | <li /><a onclick="return change_page('attributions', 1)" href="attributions">Project attribution</a> |
− | <li /><a href="">Team poster</a> | + | <li /><a onclick="return change_page('posters', 1)" href="posters">Team poster</a> |
− | <li / | + | <li />Team presentation |
− | <li /><a href="">Safety forms</a> | + | <li /><a target=_BLANK href="https://2017.igem.org/Safety/Final_Safety_Form?team_id=2351" class="url_external">Safety forms</a> |
</ul> | </ul> | ||
</td> | </td> | ||
<td width="350"> | <td width="350"> | ||
<ul> | <ul> | ||
− | <li /><a href="">Judging form</a> | + | <li /><a target=_BLANK href="https://igem.org/2017_Judging_Form?id=2351" class="url_external">Judging form</a> |
− | <li /><a href="">Registry part pages</a> | + | <li /><a onclick="return change_page('basic_part', 1)" href="basic_part">Registry part pages</a> |
− | <li /><a href="">Sample submission</a> | + | <li /><a target=_BLANK href="http://parts.igem.org/cgi/dna_transfer/batch_list.cgi?group_id=2850" class="url_external">Sample submission</a> |
− | <li /><a | + | <li /><a onclick="return change_page('interlab-study', 1)" href="interlab-study">Contribution to InterLab Study</a> |
− | + | ||
</ul> | </ul> | ||
</td> | </td> | ||
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<div style="float: left; width: 100%; margin: 0;"> | <div style="float: left; width: 100%; margin: 0;"> | ||
<b>Validate functionality of BioBrick</b><br> | <b>Validate functionality of BioBrick</b><br> | ||
− | + | BioBrick Part <a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351012" class="url_external">BBa_K2351012</a> (secreted Cpf1) has been validated. This BioBrick is very special since our team successfully secreted CRISPR-associated proteins from HEK293 cells. | |
− | + | ||
To our knowledge, this is the first time that Cpf1 has ever been expressed outside of the cell. | To our knowledge, this is the first time that Cpf1 has ever been expressed outside of the cell. | ||
+ | <br><br> | ||
+ | › <a onclick="return change_page('basic_part', 1)" href="basic_part">View submitted parts.</a> | ||
</div> | </div> | ||
<div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | <div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | ||
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Our team collaborated with the Wageningen team to obtain independent validations of both our and their biobricks. In addition, we worked with the Rathenau Institute and the RIVM on our Human Practices and safety considerations. | Our team collaborated with the Wageningen team to obtain independent validations of both our and their biobricks. In addition, we worked with the Rathenau Institute and the RIVM on our Human Practices and safety considerations. | ||
<br><br> | <br><br> | ||
− | Furthermore, we contributed a postcard design to the Düsseldorf Cologne postcards campaign. Although these are not, strictly speaking, collaborations, we have attended several meet-ups with Dutch and other European iGEM teams to exchange ideas. An overview of our collaboration efforts can be found on the | + | Furthermore, we contributed a postcard design to the Düsseldorf Cologne postcards campaign. Although these are not, strictly speaking, collaborations, we have attended several meet-ups with Dutch and other European iGEM teams to exchange ideas. An overview of our collaboration efforts can be found on the <a onclick="return change_page('collaborations', 1)" href="collaborations">Collaborations page</a>. |
</div> | </div> | ||
<div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | <div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | ||
<b>Human Practices</b><br> | <b>Human Practices</b><br> | ||
− | Our team has contacted professionals from many different backgrounds to find the setting wherein the OUTCASST system would be of most use and to identify where the requirements of the intended application affect the design of our tool. Through a series of interviews with several experts, we came to the conclusion that our tool would be most useful in diagnostics and pathogen detection in particular. By talking to a representative from ‘Doctors without borders’ and a parasitology expert, we discovered that Chagas disease is a neglected tropical disease with a large impact, yet a good diagnostic tool for this disease is still missing. Therefore, we chose to focus on Chagas disease in the further design of our tool. ( | + | Our team has contacted professionals from many different backgrounds to find the setting wherein the OUTCASST system would be of most use and to identify where the requirements of the intended application affect the design of our tool. Through a series of interviews with several experts, we came to the conclusion that our tool would be most useful in diagnostics and pathogen detection in particular. By talking to a representative from ‘Doctors without borders’ and a parasitology expert, we discovered that Chagas disease is a neglected tropical disease with a large impact, yet a good diagnostic tool for this disease is still missing. Therefore, we chose to focus on Chagas disease in the further design of our tool. Read more about our <a onclick="return change_page('stakeholders', 1)" href="stakeholders">end users</a>. |
<br><br> | <br><br> | ||
− | Besides the interviews to find the focus of our project, we also worked on outreach. In collaboration with ‘De Kennis van Nu’, a Dutch platform that brings different scientific themes to the general public, we made videos and wrote blogs about synthetic biology, the iGEM competition, lab safety, tropical diseases and our project. To raise more awareness for the competition and our project within our university, we wrote articles in several student magazines. ( | + | Besides the interviews to find the focus of our project, we also worked on outreach. In collaboration with ‘De Kennis van Nu’, a Dutch platform that brings different scientific themes to the general public, we made videos and wrote blogs about synthetic biology, the iGEM competition, lab safety, tropical diseases and our project. To raise more awareness for the competition and our project within our university, we wrote articles in several student magazines. Read more about our <a onclick="return change_page('outreach', 1)" href="outreach">outreach</a>. |
</div> | </div> | ||
</div> | </div> | ||
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<div style="float: left; width: 100%; margin: 0;"> | <div style="float: left; width: 100%; margin: 0;"> | ||
<b>Integrated Human Practices</b><br> | <b>Integrated Human Practices</b><br> | ||
− | We incorporated the feedback we got from various professionals into the design of our final product. Not only did the information we gained make us choose for Chagas disease as the focus of our project, it also made us think about the way our system could be used on location. Through our conversations with specialists, we realized the importance of simplicity in use and resistance to varying temperatures and humidity. Only tools which take these factors into account can be used without a need for further lab equipment or trained personnel. Furthermore, with safety considerations in mind, we designed our device to keep our system separated from the outside world, making it impossible for the GMO’s to escape into the environment. ( | + | We incorporated the feedback we got from various professionals into the design of our final product. Not only did the information we gained make us choose for Chagas disease as the focus of our project, it also made us think about the way our system could be used on location. Through our conversations with specialists, we realized the importance of simplicity in use and resistance to varying temperatures and humidity. Only tools which take these factors into account can be used without a need for further lab equipment or trained personnel. Furthermore, with safety considerations in mind, we designed our device to keep our system separated from the outside world, making it impossible for the GMO’s to escape into the environment. Read more about our <a onclick="return change_page('product-design', 1)" href="product-design">design considerations</a>. |
</div> | </div> | ||
<div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | <div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | ||
<b>Model your project</b><br> | <b>Model your project</b><br> | ||
− | At the same time, our team worked on modeling our system. They first summarized the kinetics of our fusion proteins in a network of reactions. With this reaction network, we demonstrated that the system contains negative feedback on its own sensitivity and give some suggestions on how to alleviate that problem with additional bio-circuitry components. In addition, we show that the precision of the system may be increased by usage of a weaker protease. We subsequently used ODE reaction equations to test if differences in substrate affinity or protein production rates can alleviate sensitivity problems and if so, how. | + | At the same time, our team worked on modeling our system. They first summarized the kinetics of our fusion proteins in a network of reactions. With this reaction network, we demonstrated that the system contains negative feedback on its own sensitivity and give some suggestions on how to alleviate that problem with additional bio-circuitry components. In addition, we show that the precision of the system may be increased by usage of a weaker protease. We subsequently used ODE reaction equations to test if differences in substrate affinity or protein production rates can alleviate sensitivity problems and if so, how. Read more about our <a onclick="return change_page('modeling-and-mathematics', 1)" href="modeling-and-mathematics">modeling efforts</a>. |
+ | </div> | ||
+ | <div style="float: left; width: 100%; margin: 0; margin-top: 20px;"> | ||
+ | <b>Improve a previous part</b><br> | ||
+ | The part we improved is <a target=_BLANK href="http://parts.igem.org/Part:BBa_K1774001" class="url_external">BBa_K1774001</a>. This part is <i>S. pyogenes</i> Cas9, submitted by the University of Hong Kong 2015 iGEM team. To improve this part we did three things: codon optimize it for mammalian cells, add a signal sequence for secretion from mammalian cells and add a His-tag. Modifying the Cas9 protein in this way makes it possible to get it produced and secreted from mammalian cells instead of bacteria. It can then be easily isolated and purified using the His-tag. Isolating the protein from mammalian cells instead of bacteria makes this Cas9 more suitable for clinical applications in humans or human cells. We therefore think this version of S. pyogenes Cas9 has added value in future medicine and thus is an improvement over the bacterial-produced version of <i>S. pyogenes</i> Cas9. | ||
+ | <br><br> | ||
+ | The improved part was submitted as <a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351013" class="url_external">BBa_K2351013</a>. | ||
+ | <br><br> | ||
+ | › <a onclick="return change_page('basic_part', 1)" href="basic_part">View all submitted parts.</a> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="page-heading">Attribution and acknowledgement</div> | <div class="page-heading">Attribution and acknowledgement</div> | ||
− | The Utrecht iGEM team performed most of the experiments, funding, PR | + | The Utrecht iGEM team performed most of the experiments, funding, PR, human practices and other tasks on their own. All additional help is acknowledged below. |
+ | For a breakdown of the contribution of individual team members, see the <a onclick="return change_page('team', 1)" href="team">Team page</a>. | ||
<br><br> | <br><br> | ||
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<br><br> | <br><br> | ||
Thank you! | Thank you! | ||
+ | </script> | ||
+ | |||
+ | <script id="page-basic_part" type="text/template"> | ||
+ | <div class="page-heading">BioBricks - Basic Parts</div> | ||
+ | |||
+ | This page contains an overview of all the basic BioBricks we created for our project.<br> | ||
+ | Interested in our composite parts? You can find them <a onclick="return change_page('composite_part', 1)" href="composite_part">here</a>. | ||
+ | |||
+ | <br><br> | ||
+ | |||
+ | <table class="biobricktable" style="width: 100%; font-size: 12px; text-align: left;"> | ||
+ | <tr style="font-size: 15px; font-weight: bold;"> | ||
+ | <td width="150">Number</td> | ||
+ | <td width="200">Name</td> | ||
+ | <td>Short description</td> | ||
+ | </tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351000" class="url_external">BBa_K2351000</a></td><td>Ig lambda-2 chain V region signal sequence</td><td>Allows for proteins to be secreted</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351001" class="url_external">BBa_K2351001</a></td><td><b>Best basic part:</b> dAsCpf1</td><td>DNA and gRNA binding properties are maintained but no endonuclease activity is exhibited.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351002" class="url_external">BBa_K2351002</a></td><td>nCas9 </td><td>A cas9 variant without first Methionine and without stopcodon, such that it can be used as a fusion protein component that contains illegal restriction sites.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351003" class="url_external">BBa_K2351003</a></td><td>nCpf1</td><td>A cpf1 variant without first Methionine and without stopcodon, such that it can be used as a fusion protein component that contains illegal restriction sites.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351004" class="url_external">BBa_K2351004</a></td><td>Glycine [3x] linked histidine[6x] tag</td><td>Basic histidine tag with preceding glycine linker.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351007" class="url_external">BBa_K2351007</a></td><td>nCas9 </td><td>A cas9 variant without first Methionine and without stopcodon, such that it can be used as a fusion protein component.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351008" class="url_external">BBa_K2351008</a></td><td>nCpf1</td><td>A cpf1 variant without first Methionine and without stopcodon, such that it can be used as a fusion protein component.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351011" class="url_external">BBa_K2351011</a></td><td>dCpf1</td><td>DNA and gRNA binding properties are maintained but no endonuclease activity is exhibited.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351012" class="url_external">BBa_K2351012</a></td><td>sCpf1</td><td>Phytobrick* // Cpf1 that will be secreted from humane cells.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351013" class="url_external">BBa_K2351013</a></td><td>dCpf1</td><td>Phytobrick*// DNA and gRNA binding properties are maintained but no endonuclease activity is exhibited.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351014" class="url_external">BBa_K2351014</a></td><td>sCas9</td><td>Phytobrick* // Cas9 that will be secreted from humane cells.</td></tr> | ||
+ | </table> | ||
+ | |||
+ | <style type="text/css"> | ||
+ | .biobricktable { border: 0 !important; margin: 0 !important; } | ||
+ | .biobricktable tr:nth-child(even) td { background: #f7f7f7; } | ||
+ | .biobricktable td { padding: 5px !important; border: 0 !important; vertical-align: middle !important; } | ||
+ | </style> | ||
+ | |||
+ | </script> | ||
+ | |||
+ | <script id="page-composite_part" type="text/template"> | ||
+ | <div class="page-heading">BioBricks - Composite Parts</div> | ||
+ | |||
+ | This page contains an overview of all the composite BioBricks we created for our project.<br> | ||
+ | Interested in our basic parts? You can find them <a onclick="return change_page('basic_part', 1)" href="basic_part">here</a>. | ||
+ | |||
+ | <br><br> | ||
+ | |||
+ | <table class="biobricktable" style="width: 100%; font-size: 12px; text-align: left;"> | ||
+ | <tr style="font-size: 15px; font-weight: bold;"> | ||
+ | <td width="150">Number</td> | ||
+ | <td width="200">Name</td> | ||
+ | <td>Short description</td> | ||
+ | </tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351005" class="url_external">BBa_K2351005</a></td><td><b>Best composite part:</b> secreted Cas9</td><td>Cas9 that will be secreted from humane cells.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351006" class="url_external">BBa_K2351006</a></td><td>Secreted Cpf1</td><td>Cpf1 that will be secreted from humane cells.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351009" class="url_external">BBa_K2351009</a></td><td>sCas9</td><td>Cas9 that will be secreted from humane cells.</td></tr> | ||
+ | <tr><td><a target=_BLANK href="http://parts.igem.org/Part:BBa_K2351010" class="url_external">BBa_K2351010</a></td><td>sCpf1</td><td>Cpf1 that will be secreted from humane cells.</td></tr> | ||
+ | </table> | ||
+ | |||
+ | <style type="text/css"> | ||
+ | .biobricktable { border: 0 !important; margin: 0 !important; } | ||
+ | .biobricktable tr:nth-child(even) td { background: #f7f7f7; } | ||
+ | .biobricktable td { padding: 5px !important; border: 0 !important; vertical-align: middle !important; } | ||
+ | </style> | ||
+ | |||
+ | </script> | ||
+ | |||
+ | <script id="page-posters" type="text/template"> | ||
+ | <div class="page-heading">Team Posters</div> | ||
+ | |||
+ | Displayed here are the posters that we used to present our team during various events. | ||
+ | |||
+ | <br><br> | ||
+ | |||
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+ | </div> | ||
+ | |||
+ | <div class="description"><a target=_BLANK href="https://static.igem.org/mediawiki/2017/5/56/UU-poster-portrait-fullsize.pdf" class="pdf">Download poster</a></div> | ||
+ | </div> | ||
+ | |||
+ | <div class="outreach-video"> | ||
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+ | </div> | ||
+ | |||
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+ | |||
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+ | |||
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+ | |||
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2: "Toolkit design solutions", | 2: "Toolkit design solutions", | ||
3: "Additional considerations", | 3: "Additional considerations", | ||
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"outreach" : {}, | "outreach" : {}, | ||
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6: "Administrative assistance", | 6: "Administrative assistance", | ||
7: "Final word" | 7: "Final word" | ||
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if(return_false) | if(return_false) | ||
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"contribution": "interlab-study", | "contribution": "interlab-study", | ||
"model": "modeling-and-mathematics", | "model": "modeling-and-mathematics", | ||
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Latest revision as of 00:24, 15 December 2017
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There are also some things in the OUTCASST toolkit that need to be changed in comparison to the experimental approach in order to prepare the system for diagnosing Chagas disease. One of these things is the use of HEK293T cells, which need a very stable environment to stay alive. In the eventual tool, we will need to use cells that are more resistant to environmental fluctuations yet still cannot survive outside of the device (Patrick van Zon and Pieter-Jaap Krijtenburg, University Medical Center of Utrecht: genome diagnostics.) We also used a fluorescence signal as output in the experiments, which requires a fluorescence microscope to analyse the test results. To avoid the need of these and other equipment, we would ideally use an output signal in the form of visible light or, more promisingly, a change of color that is visible to the naked eye. Another thing we should keep in mind is the time it takes to get the results from our test device (Marit de Wit, Doctors without Borders).
There are also technical aspects that should be considered, like the method used for lysis of the parasites in the sample. Lysis needs to occur to get free DNA, i.e. a hypotonic solution (Jaap van Hellemond, Erasmus University Medical Center Rotterdam: parasitology). If a colorant is used as reporter mechanism, we need to remove the red color of heme groups from red blood cells, too, as it would interfere with the output signal.
Lastly, we should consider the target DNA we want to use to detect the parasites. Things that require careful consideration are GC-content, which has influence on binding affinity and specificity of the guide RNA. Specificity needs to be mutation specific as a strand with different base pairs should, ideally, not activate the system (Hans Bos and Hugo Snippert, University Medical Center Utrecht: cancer research).
Toolkit design solutions
The OUTCASST toolkit has a closed box design, wherein all the components to perform the test are present in distinct compartments, separated by seals. These seals can be broken by applying pressure on them.As was stated earlier, a lot of variables need to be kept constant to keep the HEK293T cells alive. Because of this, it is not feasible to use these cells in our design. Instead, we opt to use air-dried cells from the anhydrobiotic insect, Polypelidum vanderplanki, which can be stored at room temperature for 251 days and can restart proliferating again after rehydration 1. This way the shelf life of our tool can also be prolonged. To prevent the risk of our GMO getting out in the environment, several mechanisms and kill-switches will be incorporated in the cells, so they can only survive in our closed box system, in their resurgent state. This can be done by manipulating the metabolism, so that the cells can’t produce a crucial substance for survival, e.g. an amino acid, which will be added in the toolkit medium. In case the cells get out of the toolkit, they will die because of the absence of the crucial substance.
Rehydration can be done with a suitable medium. This has to be done one hour before use. The seal between the dried insect cells and the medium can be broken to pump the medium manually to the cells. After rehydration, the medium can be manually pumped to the waste compartment.
The next step is to add the two guide RNA’s to the revived cells. The gRNA’s are present in the design as dry powder to prevent premature degradation. This time, two seals need to be broken. First, the gRNA needs to be dissolved with the contents of another medium compartment. Then the medium with gRNA can be pumped to the cells where they will bind to dCas9 and dCpf1 on the extracellular cell membrane. This process takes about 10 minutes and after that, the medium with excessive gRNA can also be pumped to the waste compartment.
These are the preparation steps before the real diagnosis can start. First off, a blood sample has to be taken from a patient that might be infected with Chagas disease. To prevent the blood from clotting, heparin or EDTA can be added to the sample. The blood sample can then be introduced to the tool, after which the device needs to be sealed. To get access to the parasite DNA, all cells need to be lysed, including the red blood cells. This is done with a lysis buffer, a hypotonic solution.
The next step is to pump everything to a next compartment, wherein there are heme-binding compounds (such as HEBP) linked to the inside surface to decolorize the sample. Then a hypertonic resetting buffer is added to return the sample to isotonic levels, in order to prevent damage to the detector cells.
Now, the seal separating the sample from the cells can be broken and the sample can be introduced to the actual sensor. A color signal will appear after about 10 to 12 hours in case the patiënt is infected and will continue to become more visible after that.
The output signal will be a blue chromoprotein. This way, the sample color will become blue (or purple if there is still a little bit of heme in the sample) upon detection of the targeted DNA sequence.
After use, the tool should be disposed of in a safe manner preventing it to end up in the environment. Therefore, there will be a disposal guideline added in the toolkit manual. The test can be disposed of in a self-sealing bag, which can be boiled after the test is completed to minimize the risks.
Additional considerations
There are still a lot of things that should be considered to make the OUTCASST tool optimal for diagnosing Chagas disease.The first thing we still need to consider is the blood sample size needed to perform the test. From the patients aspect it would be best to take as little as possible. A smaller blood sample would also mean that the device can be made smaller, which in turn also makes the production costs for one test cheaper. However, there needs to be enough pathogen DNA in the blood sample to make sure that the test gives the right results. It would be possible to pretreat a larger sample to concentrate it before applying, increasing the chance of correct diagnosis, but this would again require skilled professionals and materials.
We have also thought about a question that was raised at the University Medical Center at the Cancer department. The question was why we wanted to express our system on the membrane of eukaryotic cells and not just express it intracellularly in bacteria. Then a blood sample could be added and the bacteria can be heat shocked to get the pathogen DNA intracellular, activating the binding of the two proteins. In this case, there would be a loss of the amplification step, since the transcription factor is then able to activate the reporter gene without a signal or cleavage of the transcription factor. Since we don’t know what the minimum amount of blood needed is, we wanted to design it in the way we can get the most signal, which is to include the amplification step. If it would prove that this amplification step is not needed, we could also just put the proteins in the tool and use a split reporter. On the other hand, the tool would not rely on use of living cells, which would make the use of our tool a whole lot safer.
We should also consider the material that the device is going to be made of. It should be of sturdy quality to prevent contamination of the environment with the device’s content. From the production perspective, the costs to produce it should be as low as possible to make the tool affordable. A main issue with costs, currently, is the production of the gRNA as it is expensive to synthesize.
We have also heard that the tool should have a low incidence of false positive and negative results and that our device should distinguish DNA strands with one different base pair. We want to take this information into account to design the target DNA. There are two possibilities from which we can choose. The first option would be to permit certain mutations in the target DNA, to prevent getting a false negative result in some cases. The second option would be to use a very conserved domain as target DNA and don’t allow any mismatches. From our perspective, we think the second option would be more suitable, since the specificity in our system is a very valuable aspect of the design. We have chosen to use the satellite DNA, which is present in the T. cruzi parasite as a 195 base pair repeat with about 100,000 copies (Aldert Bart, Academical Medical Center Amsterdam: Clinical molecular parasitologist).