http://2010.igem.org/wiki/index.php?title=Special:Contributions/Pauladamiak&feed=atom&limit=50&target=Pauladamiak&year=&month=2010.igem.org - User contributions [en]2024-03-28T10:00:52ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:CalgaryTeam:Calgary2010-10-27T22:30:51Z<p>Pauladamiak: </p>
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<p class="tabText" style="padding-left:10px;">What new dangers and unintended consequences will synthetic biology pose to us in the future? iGEM Calgary travels to Defence Research and Development Canada Suffield, a major Canadian Military Research facility that specializes in chemo-biological threats. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Suffield">Read more...</a></p><br />
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<p class="tabText" style="padding-left:10px;">The University of Calgary, Alberta, and Lethbridge iGEM teams met in Lethbridge at a conference put on by Alberta Innovates Technology Futures to learn important aspects of iGEM. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Conferences#lethbridge">Read more...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Have you ever seen an aggregate protein dance? Look out for iGEM Calgary’s rambunctious mascot “Protein Man” at the Jamboree promoting proper protein expression. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Extras/Protein_Man">Learn more about him...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Team Calgary brings iGEM awareness to the community. The iGEM bake sale was a great success, both in selling cupcakes and educating customers about our team. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community">See what else we've been doing...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Alberta’s very own Jamboree. The three Alberta iGEM teams, the Universities of Alberta, Calgary, and Lethbridge meet to perform a practice project presentation to each other. Experts within their field are also present to give teams challenging questions, and then meet with each team to suggest improvements. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Conferences#agem">Read more...</a></p><br />
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<p>Our modelling project consists of two components: a mathematical model done in MATLAB and an animation done in Autodesk <i>Maya</i>. We hope to model the formation of inclusion bodies, which are aggregations of misfolded protein that can occur within cells. Using <i>Maya</i>, we also hope to visually show other students one of the proposed aggregation mechanisms described in the literature.<br />
<a href="https://2010.igem.org/Team:Calgary/Modelling">Read more here...</a></p><br />
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<p>Our outreach project included educating highschool students about iGEM. Outreach also entailed blog entries and podcast about synthetic life, iGEM and open source as well as Genetically modified foods. The blog entry, podcasts and high school presentations allowed the iGEM students to spread knowledge about iGEM to the general public and to potential future scientists. <a href="https://2010.igem.org/Team:Calgary/Community">Read more here...</a></p><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Project"><img class="projImg" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/MainPageWetlabIcon.png"></img></a></td><br />
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<h3>Wetlab</h3><br />
<p>You’re stressing me out! This year, in the wetlab, we have been designing a reporter system to detect problems in protein expression. Our system uses a visual output to allow ppecificy in determing which step in protein expression the problem is ccuring at</p><br />
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<h3>Modelling</h3><br />
<p>Our modeling project has focused on modeling and simultaing the formation of inclusion bdes, aggregates of protein within the cell, exploring the factors that lead to heri ormation</p><br />
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<h3>Community</h3><br />
<p>Our human practices section focused on exploring ethical issues in synthetic biology through an ethics paper as well as a podcast focusing on a few key issues. We also presented our project at a variety of high schools and research symposiums.</p><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:CalgaryTeam:Calgary2010-10-27T22:30:13Z<p>Pauladamiak: </p>
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<p class="tabText" style="padding-left:10px;">What new dangers and unintended consequences will synthetic biology pose to us in the future? iGEM Calgary travels to Defence Research and Development Canada Suffield, a major Canadian Military Research facility that specializes in chemo-biological threats. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Suffield">Read more...</a></p><br />
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<p class="tabText" style="padding-left:10px;">The University of Calgary, Alberta, and Lethbridge iGEM teams met in Lethbridge at a conference put on by Alberta Innovates Technology Futures to learn important aspects of iGEM. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Conferences#lethbridge">Read more...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Have you ever seen an aggregate protein dance? Look out for iGEM Calgary’s rambunctious mascot “Protein Man” at the Jamboree promoting proper protein expression. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Extras/Protein_Man">Learn more about him...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Team Calgary brings iGEM awareness to the community. The iGEM bake sale was a great success, both in selling cupcakes and educating customers about our team. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community">See what else we've been doing...</a></p><br />
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<p class="tabText" style="padding-left:10px;">Alberta’s very own Jamboree. The three Alberta iGEM teams, the Universities of Alberta, Calgary, and Lethbridge meet to perform a practice project presentation to each other. Experts within their field are also present to give teams challenging questions, and then meet with each team to suggest improvements. <a class="tabLink" href="https://2010.igem.org/Team:Calgary/Community/Conferences#agem">Read more...</a></p><br />
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<p>Our modelling project consists of two components: a mathematical model done in MATLAB and an animation done in Autodesk <i>Maya</i>. We hope to model the formation of inclusion bodies, which are aggregations of misfolded protein that can occur within cells. Using <i>Maya</i>, we also hope to visually show other students one of the proposed aggregation mechanisms described in the literature.<br />
<a href="https://2010.igem.org/Team:Calgary/Modelling">Read more here...</a></p><br />
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<p>Our outreach project included educating highschool students about iGEM. Outreach also entailed blog entries and podcast about synthetic life, iGEM and open source as well as Genetically modified foods. The blog entry, podcasts and high school presentations allowed the iGEM students to spread knowledge about iGEM to the general public and to potential future scientists. <a href="https://2010.igem.org/Team:Calgary/Community">Read more here...</a></p><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Project"><img class="projImg" src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/MainPageWetlabIcon.png"></img></a></td><br />
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<h3>Wetlab</h3><br />
<p>You’re stressing me out! This year, in the wetlab, we have been designing a reporter system to detect problems in protein expression. Our system uses a visual output to allow ppecificy in determing which step in protein expression the problem is ccuring at</p><br />
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<h3>Modelling</h3><br />
<p>Our modeling project has focused on modeling and simultaing the formation of inclusion bdes, aggregates of protein within the cell, exploring the factors that lead to heri ormation</p><br />
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<h3>Community</h3><br />
<p>Our human practices section focused on exploring ethical issues in synthetic biology through an ethics paper as well as a podcast focusing on a few key issues. We also presented our project at a variety of high schools and research symposiums.</p><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#bioalb"><img src="https://static.igem.org/mediawiki/2008/7/7b/Bioalberta.jpeg"></img></a></td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#aihs"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/AlHSLogo.png"></img></a></td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Sponsors#corning"><img src="http://www.ysbl.york.ac.uk/fbld/2010/Corning100logo.jpg"></img></a></td><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Modelling/MATLABTeam:Calgary/Modelling/MATLAB2010-10-27T22:00:32Z<p>Pauladamiak: </p>
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<h1>Modelling</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/MATLAB">MATLAB Models</a><br />
<ul><br />
<li><a href="#MATLAB">Matrix Laboratory Software</a></li><br />
<li><a href="#Protein">Protein production abstraction</a></li><br />
<li><a href="#Relationships">Relationship equations</a></li><br />
<li><a href="#Factors">Factors under investigation</a></li><br />
<li><a href="#Results">Result cases</a></li><br />
<li><a href="#Conclusions">Conclusions</a></li><br />
<li><a href="#Future">Future directions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/Maya">Maya Animations</a></li><br />
<ul><br />
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</div><br />
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<div class="mainbody"><br />
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<span id="bodytitle"><h1 id="MATLAB">MATLAB</h1></span><br />
<h2 style="color:#0066CC">Matrix Laboratory Software </h2><br />
<p>The protein production simulation was produced using the Matrix Laboratory software, MATLAB produced by MathWorks. Specifically this project uses the Simbiology application, which is a collection of computational tools for simulating biological processes. The power of this tool lies in its ability to build multi species and reaction models, then simulate how the species will interact.<br />
</p><br /><br />
<br />
<br />
<h2 id="Protein" style="color:#0066CC">Protein production abstraction</h2><br />
<p>Our proposed model relies on an abstraction of the process of protein production, taking into account the formation of incorrectly folded intermediates and the presence of aggregated misfolded protein. Our proposed model is shown below</p> <br />
<table><br />
<tr><td valign="top"><img style="margin-right:5px;" src="https://static.igem.org/mediawiki/2010/2/24/Model_flow_chart.png" title="MATLAB model network" /></td><br />
<td align="justify"><p><b>Figure 1.</b> The proposed path consists of a number of species. The first one identified is "natal peptide" this species represents the initial amount of amino acid chain being produced by the ribosome. It is the "starting" amount of potential protein/misfolded protein/inclusion body present in the system.</p><br />
<p>The second species identified is the "unstable protein" this references proteins that have not reached their fully stable conformations yet or have been destabilized by environmental conditions. Unstable protein has the potential to be degraded by cellular proteases which results in the "degraded protein" species. Additionally unstable proteins have the potential to clump together to form the "inclusion body" species. The inclusion body species is also degraded by proteases so the degraded protein species is also a potential result.</p><br />
<p>The final species is the stable functional protein form. This stable form is also degraded by proteases but in very very small amounts</p> <br />
</td></tr><br />
</table><br /><br />
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<br />
<h2 id="Relationships" style="color:#0066CC">Relationship equations</h2><br />
<p>In order to begin investigating the factors that may affect the final state of the natal peptide it first becomes important to define how each of the species in the system interact. This is where the MATLAB software becomes useful. The proposed model pathway can be represented in the MATLAB Simbiology Toolbox.</p><br />
<table><br />
<tr><br />
<td valign="top" align="justify"><b>Figure 2.</b> Each of the blue circles correspond to the species identified in the previous section, while the yellow circles define the reaction that occurs between the two species. The Simbiology software uses these defined reactions to determine the amounts of each species present in the system as the species interact over a period of time. The results of the simulation are described in a later section<br />
</td><br />
<td><br />
<img style="margin-left:5px; width:409px; height:347px;" src="https://static.igem.org/mediawiki/2010/1/12/Matlab_network.png" title="MATLAB Diagram View" /><br />
</td><br />
</tr><br />
</table><br /><br />
<p>The reactions present in the system are described by the following equations</p><br /><br />
<table><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>natal peptide -> unstable protein</em></td><br />
<td><p>This reaction is an irreversible reaction where all of the natal peptide present becomes unstable protein with a rate constant of 1. This reflects the assumption that all of the initial amino acid sequence will at some point be present as unstable protein capable of being degraded or forming inclusion bodies</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein + inclusion body <-> 2 inclusion body</em></td><br />
<td><p>This reaction is a reversible reaction that defines how unstable proteins form into inclusion bodies. The simulation assumes that there is a baseline very small concentration of inclusion body present. This base value may interact with unstable protein to form more inclusion bodies resulting in a positive feed back that begins to form higher concentrations of inclusion bodies. The rate constant at which this process occurs is dependent on a number of factors discussed in a later section. Inclusion body formation represents a potential outcome of the protein expression system</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>unstable protein -> degraded protein</em></td><br />
<td><p>This reaction represents unstable protein being degraded by proteases. This reaction follows the Henri-Michaelis-Menten equilibrium process. In this process there is a pseudo equilibrium present that is determined to be one way, as degraded protein does not return to its pre degraded state. The constant for this reaction is a set value that is relative to the particular protease. For simplicity a single value was selected to represent this impact. Once the unstable protein has been degraded it is removed from the simulation system and can not form inclusion bodies or functional proteins. As a result degraded protein represents one of the potential "outcomes" of a protein expression experiment</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>inclusion body -> degraded protein</em></td><br />
<td><p>Our system assumes that inclusion bodies are degraded by the same process as unstable proteins, but that the rate of degradation is slower than that of a single unstable protein. This is proposed to be the result of the protease being unable to access individual proteins of the inclusion body for degradation. As a result the process is considerably slower than the degradation of straight unstable proteins. The difference is reflected in the rate constant for each of the two reactions</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein <-> functional protein</em></td><br />
<td><p>This reaction represents the process of the unstable protein becoming stable functional protein. The reaction is reversible as different environmental conditions, discussed later on, can determine whether or not the protein is in a stable state or unstable state.</p><p></p></td><br />
</tr></table><br />
<br /><br />
<br />
<br />
<h2 id="Factors" style="color:#0066CC">Factors under investigation</h2><br />
<p> For the purpose of our model there were two categories of factors that we investigated. These categories are Environmental factors and sequence factors. It is important to note that these divisions are arbitrary and don't necessarily exist in reality. Specifically these categories were created for the convenience of organizing the factors being investigated and determining how to best evaluate their impact.</p><br />
<h3>Environmental factors</h3><br />
<p>The collection of factors refers to those features of the cells environment that will have an impact on the stability of proteins being produced. When we use the term environment we are referring to both the external temperature at which the cell is growing, the pH of its environment and the concentration of protein within cellular compartments. These factors are not determined by features of the protein being produced but will still affect the likelihood of inclusion body formation and/or protein instability.<br />
</p><br />
<h4 style="color:#003366">Defining environmental impact on protein stability</h4><br />
<p>We have assumed that environmental conditions affect inclusion body formation by altering the equilibrium of the following equation:</p><br />
<br />
<div style="width:450px; margin-left:auto; margin-right:auto;"><b>Functional Protein <-> Unstable Protein <-> Inclusion Body </b></div><br /><br />
<br />
<p>A publication by Brandt et al supports this concept as they showed that isolated protein in a solution can be converted back and forth from its stable form and inclusion body form based on temperature and pH. Specifically that high temperatures and strongly basic pH will encourage the formation of inclusion bodies. This means that equilibrium constant increases as the formation of inclusion bodies has become more favourable</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>From this information we have assumed that altering the environmental conditions of temperature, pH and protein concentration will have a quantifiable effect on the rate constants for the above equation. Currently the exact impact of these factors hasn't been determined. The results section only describes rate constants determined for convenience based on qualitative understanding of the processes.<br />
</p><br />
<br />
<h3>Sequence dependent factors</h3> <br />
<p>For these factors we have tried to look at the features of the mRNA and amino acid sequence that could impact the likelihood of inclusion body formation. As a general rule sequence factors were selected based on how the particular feature affects the time taken for the sequence to reach its fully folded stable confirmation. The reason for this based on the assumption that if the protein has more intermediate stages or is more thermodynamically stable in a non folded confirmation then there is more time for the unstable proteins to interact and begin the formation of inclusion bodies.</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>Sequence features such as scarce amino acids ( Tryptohphan ), mRNA structural features that inhibit translation time through the ribosome and the ratio of hydrophobic amino acids to charged amino acids all have a quantifiable effect on the rate constant at which unstable protein becomes stable protein.</p><br />
<br />
<p>This evidence has been indirectly supported from the literature and discussions with researchers in the field. However at this point the precise impact of these factors is still under investigation. As such for the results section, hypothesized relationships alone have been used.</p><br /><br />
<br />
<br />
<br />
<br />
<h2 id="Results" style="color:#0066CC">Result cases</h2><br />
<p>The result cases represent the preliminary testing of the model to see if the model simulation can be used to analyze the protein production process under different conditions. The rate constants used for each of the cases were selected for convenience in order to determine if relevant results could be obtained. This means that the rate constants used in the models do not directly correspond to biological data. However the relationships between the different rate constants are representative of biological data. It is important to note that this data is very preliminary and only demonstrates that the modelling approach we have taken can be used to investigate the factors affecting protein misfolding.<br />
</p><br />
<br />
<h3>Result Case 1: Successful expression of stable protein</h3><br />
<p>In this case the initial amount of peptide produced is very stable and the equilibrium favours the fast formation of stable correctly folded protein ( red line). Unstable protein is still produced in this scenario, but is quickly degraded by and does not form inclusion bodies. Additionally the unstable protein is not exposing significant hydrophobic amino acids. This also helps prevent the formation of inclusion bodies.</p><br />
<img style="margin-left:-15px; width:682px; height:394px" src="https://static.igem.org/mediawiki/2010/b/bf/Stable_production.png" /><br /><br />
<table><br />
<p><b>Figure 3. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 M ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>There is still a peak in unstable protein, as this species will still be present. But this amount will be degraded quickly.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the natal peptide is quickly driven towards stable protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>In this scenario very few inclusion bodies are produced as there is not enough unstable protein present to induce nucleation</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h3>Result Case 2: High hydrophobic to charged amino acid ratio with a protein having many unstable intermediates </h3><br />
<p><br />
In this case the produced peptide is highly unstable and is composed of significantly more hydrophobic amino acids than charged amino acids. This indicates that in the unstable form a significant amount of the exposed amino acids will be hydrophobic. This state of unstable protein will strongly drive towards inclusion body formation.<br />
</p><br />
<img style="margin-left:-15px; width:682px; height:394px" src="https://static.igem.org/mediawiki/2010/a/a7/Highly_unstable.png" /><br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>A high hydropathy value increases the time required for the protein to fold into its correct shape. This means more unstable protein will be present in the equilibrium. The green line on the graph represents this value. The peak on the graph represents the point of nucleation whereby the concentration of unstable protein reaches a peak that rapidly increases the drive to inclusion bodies.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>The increased hydropathy content decreases the presence of functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line )</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<br />
<br />
<h3>Result Case 3: Over produced protein at high temperature</h3><br />
<p>In this scenario the protein is highly stable and drives strongly towards it’s stable functional conformation. However the over production of the peptide means there are high concentrations of unstable protein present, and the higher temperature increases the kinetic movement of the unstable protein. This causes the unstable proteins to collide more frequently and form inclusion bodies before they have a chance to become stable protein. Since the protein is highly stable the nucleation point, the peak of the green line, occurs at a higher concentration and greater time value than that of the unstable protein in the previous case.</p><br /><br />
<img style="margin-left:-15px; width:682px; height:394px" src="https://static.igem.org/mediawiki/2010/3/31/Hightemp_stable.png" /><br /> <br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>The highly stable protein produced in this scenario spends very little time in the unstable form. It is quickly converted to stable protein or driven into an inclusion body. In this scenario the concentration and temperature causes the equilibrium to favor inclusion bodies over the functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the functional protein is very stable and favored. However the concentration of unstable intermediate causes the intermediate proteins to be caught up in inclusion bodies prior to formation of correctly folded protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line ). This point is reached more slowly than in the previous scenario as the protein is more stable, and a certain concentration must be met before nucleation occurs.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h2 id="Conclusions" style="color:#0066CC">Conclusions</h2><br />
<p> <br />
There are a number of immediate problems with this model approach that affect the accuracy of the results. The first and most significant is that the values for the amounts of species and the kinetic constants were selected for convenience. This means they were selected such that their relative relationships would allow us to determine if the model could be made to match the behaviour seen in the literature. As a result the the outputs of the simulation can not be taken as "real" because they do not use true values. The second major issue is the equilibrium equations proposed for each of the reactions are abstractions and may not be the best ways of relating the species. The third major issue is that there aren't any clearly defined relationships between each of the factors being investigated and misfiling. Therefore the relationship between factors such as temperature and sequence features such as hydrophobic/charged amino acids are accounted for in a qualitative way only.<br />
</p><br />
<p>Accounting for these caveats this model was still a success in a number of ways. The results demonstrate that the MATLAB Simbiology software can be used to simulate the process of inclusion body formation. The graphs obtained match closely, albeit in a qualitative way, the process of inclusion body formation as it is described in the literature. Lastly, this approach has provided us with a framework that can be used to study the factors affecting protein misfolding and aggregation. <br />
</p><br /><br />
<br />
<h2 id="Future" style="color:#0066CC">Future directions</h2><br />
<p>The most necessary future direction is to find a "test" protein and apply the principles of the model to determine if the simulation results match with literature results. If this can be shown more test proteins can be evaluated with the model and the model can be made more general. The second future direction is to explore the concept of "cut off" values. From the equilibrium graphs in the results section it is clear that there is some amount of inclusion body present, some amount of unstable protein and some amount of stable functional protein. This implies that there will always be a ratio between the three different species and also that even when inclusion body is present some functional protein will be too. A cut off value would be the ratio of functional protein to non functional such that functional protein can still be obtained. The final future direction is to develop a way to account for the four different categories of inclusion bodies that are seen in the literature values. <br />
</p><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Modelling/MATLABTeam:Calgary/Modelling/MATLAB2010-10-27T21:58:41Z<p>Pauladamiak: </p>
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<h1>Modelling</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/MATLAB">MATLAB Models</a><br />
<ul><br />
<li><a href="#MATLAB">Matrix Laboratory Software</a></li><br />
<li><a href="#Protein">Protein production abstraction</a></li><br />
<li><a href="#Relationships">Relationship equations</a></li><br />
<li><a href="#Factors">Factors under investigation</a></li><br />
<li><a href="#Results">Result cases</a></li><br />
<li><a href="#Conclusions">Conclusions</a></li><br />
<li><a href="#Future">Future directions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/Maya">Maya Animations</a></li><br />
<ul><br />
<br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1 id="MATLAB">MATLAB</h1></span><br />
<h2 style="color:#0066CC">Matrix Laboratory Software </h2><br />
<p>The protein production simulation was produced using the Matrix Laboratory software, MATLAB produced by MathWorks. Specifically this project uses the Simbiology application, which is a collection of computational tools for simulating biological processes. The power of this tool lies in its ability to build multi species and reaction models, then simulate how the species will interact.<br />
</p><br /><br />
<br />
<br />
<h2 id="Protein" style="color:#0066CC">Protein production abstraction</h2><br />
<p>Our proposed model relies on an abstraction of the process of protein production, taking into account the formation of incorrectly folded intermediates and the presence of aggregated misfolded protein. Our proposed model is shown below</p> <br />
<table><br />
<tr><td valign="top"><img style="margin-right:5px;" src="https://static.igem.org/mediawiki/2010/2/24/Model_flow_chart.png" title="MATLAB model network" /></td><br />
<td align="justify"><p><b>Figure 1.</b> The proposed path consists of a number of species. The first one identified is "natal peptide" this species represents the initial amount of amino acid chain being produced by the ribosome. It is the "starting" amount of potential protein/misfolded protein/inclusion body present in the system.</p><br />
<p>The second species identified is the "unstable protein" this references proteins that have not reached their fully stable conformations yet or have been destabilized by environmental conditions. Unstable protein has the potential to be degraded by cellular proteases which results in the "degraded protein" species. Additionally unstable proteins have the potential to clump together to form the "inclusion body" species. The inclusion body species is also degraded by proteases so the degraded protein species is also a potential result.</p><br />
<p>The final species is the stable functional protein form. This stable form is also degraded by proteases but in very very small amounts</p> <br />
</td></tr><br />
</table><br /><br />
<br />
<br />
<h2 id="Relationships" style="color:#0066CC">Relationship equations</h2><br />
<p>In order to begin investigating the factors that may affect the final state of the natal peptide it first becomes important to define how each of the species in the system interact. This is where the MATLAB software becomes useful. The proposed model pathway can be represented in the MATLAB Simbiology Toolbox.</p><br />
<table><br />
<tr><br />
<td valign="top" align="justify"><b>Figure 2.</b> Each of the blue circles correspond to the species identified in the previous section, while the yellow circles define the reaction that occurs between the two species. The Simbiology software uses these defined reactions to determine the amounts of each species present in the system as the species interact over a period of time. The results of the simulation are described in a later section<br />
</td><br />
<td><br />
<img style="margin-left:5px; width:409px; height:347px;" src="https://static.igem.org/mediawiki/2010/1/12/Matlab_network.png" title="MATLAB Diagram View" /><br />
</td><br />
</tr><br />
</table><br /><br />
<p>The reactions present in the system are described by the following equations</p><br /><br />
<table><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>natal peptide -> unstable protein</em></td><br />
<td><p>This reaction is an irreversible reaction where all of the natal peptide present becomes unstable protein with a rate constant of 1. This reflects the assumption that all of the initial amino acid sequence will at some point be present as unstable protein capable of being degraded or forming inclusion bodies</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein + inclusion body <-> 2 inclusion body</em></td><br />
<td><p>This reaction is a reversible reaction that defines how unstable proteins form into inclusion bodies. The simulation assumes that there is a baseline very small concentration of inclusion body present. This base value may interact with unstable protein to form more inclusion bodies resulting in a positive feed back that begins to form higher concentrations of inclusion bodies. The rate constant at which this process occurs is dependent on a number of factors discussed in a later section. Inclusion body formation represents a potential outcome of the protein expression system</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>unstable protein -> degraded protein</em></td><br />
<td><p>This reaction represents unstable protein being degraded by proteases. This reaction follows the Henri-Michaelis-Menten equilibrium process. In this process there is a pseudo equilibrium present that is determined to be one way, as degraded protein does not return to its pre degraded state. The constant for this reaction is a set value that is relative to the particular protease. For simplicity a single value was selected to represent this impact. Once the unstable protein has been degraded it is removed from the simulation system and can not form inclusion bodies or functional proteins. As a result degraded protein represents one of the potential "outcomes" of a protein expression experiment</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>inclusion body -> degraded protein</em></td><br />
<td><p>Our system assumes that inclusion bodies are degraded by the same process as unstable proteins, but that the rate of degradation is slower than that of a single unstable protein. This is proposed to be the result of the protease being unable to access individual proteins of the inclusion body for degradation. As a result the process is considerably slower than the degradation of straight unstable proteins. The difference is reflected in the rate constant for each of the two reactions</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein <-> functional protein</em></td><br />
<td><p>This reaction represents the process of the unstable protein becoming stable functional protein. The reaction is reversible as different environmental conditions, discussed later on, can determine whether or not the protein is in a stable state or unstable state.</p><p></p></td><br />
</tr></table><br />
<br /><br />
<br />
<br />
<h2 id="Factors" style="color:#0066CC">Factors under investigation</h2><br />
<p> For the purpose of our model there were two categories of factors that we investigated. These categories are Environmental factors and sequence factors. It is important to note that these divisions are arbitrary and don't necessarily exist in reality. Specifically these categories were created for the convenience of organizing the factors being investigated and determining how to best evaluate their impact.</p><br />
<h3>Environmental factors</h3><br />
<p>The collection of factors refers to those features of the cells environment that will have an impact on the stability of proteins being produced. When we use the term environment we are referring to both the external temperature at which the cell is growing, the pH of its environment and the concentration of protein within cellular compartments. These factors are not determined by features of the protein being produced but will still affect the likelihood of inclusion body formation and/or protein instability.<br />
</p><br />
<h4 style="color:#003366">Defining environmental impact on protein stability</h4><br />
<p>We have assumed that environmental conditions affect inclusion body formation by altering the equilibrium of the following equation:</p><br />
<br />
<div style="width:450px; margin-left:auto; margin-right:auto;"><b>Functional Protein <-> Unstable Protein <-> Inclusion Body </b></div><br /><br />
<br />
<p>A publication by Brandt et al supports this concept as they showed that isolated protein in a solution can be converted back and forth from its stable form and inclusion body form based on temperature and pH. Specifically that high temperatures and strongly basic pH will encourage the formation of inclusion bodies. This means that equilibrium constant increases as the formation of inclusion bodies has become more favourable</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>From this information we have assumed that altering the environmental conditions of temperature, pH and protein concentration will have a quantifiable effect on the rate constants for the above equation. Currently the exact impact of these factors hasn't been determined. The results section only describes rate constants determined for convenience based on qualitative understanding of the processes.<br />
</p><br />
<br />
<h3>Sequence dependent factors</h3> <br />
<p>For these factors we have tried to look at the features of the mRNA and amino acid sequence that could impact the likelihood of inclusion body formation. As a general rule sequence factors were selected based on how the particular feature affects the time taken for the sequence to reach its fully folded stable confirmation. The reason for this based on the assumption that if the protein has more intermediate stages or is more thermodynamically stable in a non folded confirmation then there is more time for the unstable proteins to interact and begin the formation of inclusion bodies.</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>Sequence features such as scarce amino acids ( Tryptohphan ), mRNA structural features that inhibit translation time through the ribosome and the ratio of hydrophobic amino acids to charged amino acids all have a quantifiable effect on the rate constant at which unstable protein becomes stable protein.</p><br />
<br />
<p>This evidence has been indirectly supported from the literature and discussions with researchers in the field. However at this point the precise impact of these factors is still under investigation. As such for the results section, hypothesized relationships alone have been used.</p><br /><br />
<br />
<br />
<br />
<br />
<h2 id="Results" style="color:#0066CC">Result cases</h2><br />
<p>The result cases represent the preliminary testing of the model to see if the model simulation can be used to analyze the protein production process under different conditions. The rate constants used for each of the cases were selected for convenience in order to determine if relevant results could be obtained. This means that the rate constants used in the models do not directly correspond to biological data. However the relationships between the different rate constants are representative of biological data. It is important to note that this data is very preliminary and only demonstrates that the modelling approach we have taken can be used to investigate the factors affecting protein misfolding.<br />
</p><br />
<br />
<h3>Result Case 1: Successful expression of stable protein</h3><br />
<p>In this case the initial amount of peptide produced is very stable and the equilibrium favours the fast formation of stable correctly folded protein ( red line). Unstable protein is still produced in this scenario, but is quickly degraded by and does not form inclusion bodies. Additionally the unstable protein is not exposing significant hydrophobic amino acids. This also helps prevent the formation of inclusion bodies.</p><br />
<img style="margin-left:-15px; width:682px; height:394px" src="https://static.igem.org/mediawiki/2010/b/bf/Stable_production.png" /><br /><br />
<table><br />
<p><b>Figure 3. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 M ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>There is still a peak in unstable protein, as this species will still be present. But this amount will be degraded quickly.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the natal peptide is quickly driven towards stable protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>In this scenario very few inclusion bodies are produced as there is not enough unstable protein present to induce nucleation</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h3>Result Case 2: High hydrophobic to charged amino acid ratio with a protein having many unstable intermediates </h3><br />
<p><br />
In this case the produced peptide is highly unstable and is composed of significantly more hydrophobic amino acids than charged amino acids. This indicates that in the unstable form a significant amount of the exposed amino acids will be hydrophobic. This state of unstable protein will strongly drive towards inclusion body formation.<br />
</p><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/a/a7/Highly_unstable.png" /><br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>A high hydropathy value increases the time required for the protein to fold into its correct shape. This means more unstable protein will be present in the equilibrium. The green line on the graph represents this value. The peak on the graph represents the point of nucleation whereby the concentration of unstable protein reaches a peak that rapidly increases the drive to inclusion bodies.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>The increased hydropathy content decreases the presence of functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line )</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<br />
<br />
<h3>Result Case 3: Over produced protein at high temperature</h3><br />
<p>In this scenario the protein is highly stable and drives strongly towards it’s stable functional conformation. However the over production of the peptide means there are high concentrations of unstable protein present, and the higher temperature increases the kinetic movement of the unstable protein. This causes the unstable proteins to collide more frequently and form inclusion bodies before they have a chance to become stable protein. Since the protein is highly stable the nucleation point, the peak of the green line, occurs at a higher concentration and greater time value than that of the unstable protein in the previous case.</p><br /><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/3/31/Hightemp_stable.png" /><br /> <br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>The highly stable protein produced in this scenario spends very little time in the unstable form. It is quickly converted to stable protein or driven into an inclusion body. In this scenario the concentration and temperature causes the equilibrium to favor inclusion bodies over the functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the functional protein is very stable and favored. However the concentration of unstable intermediate causes the intermediate proteins to be caught up in inclusion bodies prior to formation of correctly folded protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line ). This point is reached more slowly than in the previous scenario as the protein is more stable, and a certain concentration must be met before nucleation occurs.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h2 id="Conclusions" style="color:#0066CC">Conclusions</h2><br />
<p> <br />
There are a number of immediate problems with this model approach that affect the accuracy of the results. The first and most significant is that the values for the amounts of species and the kinetic constants were selected for convenience. This means they were selected such that their relative relationships would allow us to determine if the model could be made to match the behaviour seen in the literature. As a result the the outputs of the simulation can not be taken as "real" because they do not use true values. The second major issue is the equilibrium equations proposed for each of the reactions are abstractions and may not be the best ways of relating the species. The third major issue is that there aren't any clearly defined relationships between each of the factors being investigated and misfiling. Therefore the relationship between factors such as temperature and sequence features such as hydrophobic/charged amino acids are accounted for in a qualitative way only.<br />
</p><br />
<p>Accounting for these caveats this model was still a success in a number of ways. The results demonstrate that the MATLAB Simbiology software can be used to simulate the process of inclusion body formation. The graphs obtained match closely, albeit in a qualitative way, the process of inclusion body formation as it is described in the literature. Lastly, this approach has provided us with a framework that can be used to study the factors affecting protein misfolding and aggregation. <br />
</p><br /><br />
<br />
<h2 id="Future" style="color:#0066CC">Future directions</h2><br />
<p>The most necessary future direction is to find a "test" protein and apply the principles of the model to determine if the simulation results match with literature results. If this can be shown more test proteins can be evaluated with the model and the model can be made more general. The second future direction is to explore the concept of "cut off" values. From the equilibrium graphs in the results section it is clear that there is some amount of inclusion body present, some amount of unstable protein and some amount of stable functional protein. This implies that there will always be a ratio between the three different species and also that even when inclusion body is present some functional protein will be too. A cut off value would be the ratio of functional protein to non functional such that functional protein can still be obtained. The final future direction is to develop a way to account for the four different categories of inclusion bodies that are seen in the literature values. <br />
</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Modelling/MATLABTeam:Calgary/Modelling/MATLAB2010-10-27T21:57:03Z<p>Pauladamiak: </p>
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<h1>Modelling</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/MATLAB">MATLAB Models</a><br />
<ul><br />
<li><a href="#MATLAB">Matrix Laboratory Software</a></li><br />
<li><a href="#Protein">Protein production abstraction</a></li><br />
<li><a href="#Relationships">Relationship equations</a></li><br />
<li><a href="#Factors">Factors under investigation</a></li><br />
<li><a href="#Results">Result cases</a></li><br />
<li><a href="#Conclusions">Conclusions</a></li><br />
<li><a href="#Future">Future directions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/Maya">Maya Animations</a></li><br />
<ul><br />
<br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1 id="MATLAB">MATLAB</h1></span><br />
<h2 style="color:#0066CC">Matrix Laboratory Software </h2><br />
<p>The protein production simulation was produced using the Matrix Laboratory software, MATLAB produced by MathWorks. Specifically this project uses the Simbiology application, which is a collection of computational tools for simulating biological processes. The power of this tool lies in its ability to build multi species and reaction models, then simulate how the species will interact.<br />
</p><br /><br />
<br />
<br />
<h2 id="Protein" style="color:#0066CC">Protein production abstraction</h2><br />
<p>Our proposed model relies on an abstraction of the process of protein production, taking into account the formation of incorrectly folded intermediates and the presence of aggregated misfolded protein. Our proposed model is shown below</p> <br />
<table><br />
<tr><td valign="top"><img style="margin-right:5px;" src="https://static.igem.org/mediawiki/2010/2/24/Model_flow_chart.png" title="MATLAB model network" /></td><br />
<td align="justify"><p><b>Figure 1.</b> The proposed path consists of a number of species. The first one identified is "natal peptide" this species represents the initial amount of amino acid chain being produced by the ribosome. It is the "starting" amount of potential protein/misfolded protein/inclusion body present in the system.</p><br />
<p>The second species identified is the "unstable protein" this references proteins that have not reached their fully stable conformations yet or have been destabilized by environmental conditions. Unstable protein has the potential to be degraded by cellular proteases which results in the "degraded protein" species. Additionally unstable proteins have the potential to clump together to form the "inclusion body" species. The inclusion body species is also degraded by proteases so the degraded protein species is also a potential result.</p><br />
<p>The final species is the stable functional protein form. This stable form is also degraded by proteases but in very very small amounts</p> <br />
</td></tr><br />
</table><br /><br />
<br />
<br />
<h2 id="Relationships" style="color:#0066CC">Relationship equations</h2><br />
<p>In order to begin investigating the factors that may affect the final state of the natal peptide it first becomes important to define how each of the species in the system interact. This is where the MATLAB software becomes useful. The proposed model pathway can be represented in the MATLAB Simbiology Toolbox.</p><br />
<table><br />
<tr><br />
<td valign="top" align="justify"><b>Figure 2.</b> Each of the blue circles correspond to the species identified in the previous section, while the yellow circles define the reaction that occurs between the two species. The Simbiology software uses these defined reactions to determine the amounts of each species present in the system as the species interact over a period of time. The results of the simulation are described in a later section<br />
</td><br />
<td><br />
<img style="margin-left:5px; width:409px; height:347px;" src="https://static.igem.org/mediawiki/2010/1/12/Matlab_network.png" title="MATLAB Diagram View" /><br />
</td><br />
</tr><br />
</table><br /><br />
<p>The reactions present in the system are described by the following equations</p><br /><br />
<table><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>natal peptide -> unstable protein</em></td><br />
<td><p>This reaction is an irreversible reaction where all of the natal peptide present becomes unstable protein with a rate constant of 1. This reflects the assumption that all of the initial amino acid sequence will at some point be present as unstable protein capable of being degraded or forming inclusion bodies</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein + inclusion body <-> 2 inclusion body</em></td><br />
<td><p>This reaction is a reversible reaction that defines how unstable proteins form into inclusion bodies. The simulation assumes that there is a baseline very small concentration of inclusion body present. This base value may interact with unstable protein to form more inclusion bodies resulting in a positive feed back that begins to form higher concentrations of inclusion bodies. The rate constant at which this process occurs is dependent on a number of factors discussed in a later section. Inclusion body formation represents a potential outcome of the protein expression system</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>unstable protein -> degraded protein</em></td><br />
<td><p>This reaction represents unstable protein being degraded by proteases. This reaction follows the Henri-Michaelis-Menten equilibrium process. In this process there is a pseudo equilibrium present that is determined to be one way, as degraded protein does not return to its pre degraded state. The constant for this reaction is a set value that is relative to the particular protease. For simplicity a single value was selected to represent this impact. Once the unstable protein has been degraded it is removed from the simulation system and can not form inclusion bodies or functional proteins. As a result degraded protein represents one of the potential "outcomes" of a protein expression experiment</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>inclusion body -> degraded protein</em></td><br />
<td><p>Our system assumes that inclusion bodies are degraded by the same process as unstable proteins, but that the rate of degradation is slower than that of a single unstable protein. This is proposed to be the result of the protease being unable to access individual proteins of the inclusion body for degradation. As a result the process is considerably slower than the degradation of straight unstable proteins. The difference is reflected in the rate constant for each of the two reactions</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein <-> functional protein</em></td><br />
<td><p>This reaction represents the process of the unstable protein becoming stable functional protein. The reaction is reversible as different environmental conditions, discussed later on, can determine whether or not the protein is in a stable state or unstable state.</p><p></p></td><br />
</tr></table><br />
<br /><br />
<br />
<br />
<h2 id="Factors" style="color:#0066CC">Factors under investigation</h2><br />
<p> For the purpose of our model there were two categories of factors that we investigated. These categories are Environmental factors and sequence factors. It is important to note that these divisions are arbitrary and don't necessarily exist in reality. Specifically these categories were created for the convenience of organizing the factors being investigated and determining how to best evaluate their impact.</p><br />
<h3>Environmental factors</h3><br />
<p>The collection of factors refers to those features of the cells environment that will have an impact on the stability of proteins being produced. When we use the term environment we are referring to both the external temperature at which the cell is growing, the pH of its environment and the concentration of protein within cellular compartments. These factors are not determined by features of the protein being produced but will still affect the likelihood of inclusion body formation and/or protein instability.<br />
</p><br />
<h4 style="color:#003366">Defining environmental impact on protein stability</h4><br />
<p>We have assumed that environmental conditions affect inclusion body formation by altering the equilibrium of the following equation:</p><br />
<br />
<div style="width:450px; margin-left:auto; margin-right:auto;"><b>Functional Protein <-> Unstable Protein <-> Inclusion Body </b></div><br /><br />
<br />
<p>A publication by Brandt et al supports this concept as they showed that isolated protein in a solution can be converted back and forth from its stable form and inclusion body form based on temperature and pH. Specifically that high temperatures and strongly basic pH will encourage the formation of inclusion bodies. This means that equilibrium constant increases as the formation of inclusion bodies has become more favourable</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>From this information we have assumed that altering the environmental conditions of temperature, pH and protein concentration will have a quantifiable effect on the rate constants for the above equation. Currently the exact impact of these factors hasn't been determined. The results section only describes rate constants determined for convenience based on qualitative understanding of the processes.<br />
</p><br />
<br />
<h3>Sequence dependent factors</h3> <br />
<p>For these factors we have tried to look at the features of the mRNA and amino acid sequence that could impact the likelihood of inclusion body formation. As a general rule sequence factors were selected based on how the particular feature affects the time taken for the sequence to reach its fully folded stable confirmation. The reason for this based on the assumption that if the protein has more intermediate stages or is more thermodynamically stable in a non folded confirmation then there is more time for the unstable proteins to interact and begin the formation of inclusion bodies.</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>Sequence features such as scarce amino acids ( Tryptohphan ), mRNA structural features that inhibit translation time through the ribosome and the ratio of hydrophobic amino acids to charged amino acids all have a quantifiable effect on the rate constant at which unstable protein becomes stable protein.</p><br />
<br />
<p>This evidence has been indirectly supported from the literature and discussions with researchers in the field. However at this point the precise impact of these factors is still under investigation. As such for the results section, hypothesized relationships alone have been used.</p><br /><br />
<br />
<br />
<br />
<br />
<h2 id="Results" style="color:#0066CC">Result cases</h2><br />
<p>The result cases represent the preliminary testing of the model to see if the model simulation can be used to analyze the protein production process under different conditions. The rate constants used for each of the cases were selected for convenience in order to determine if relevant results could be obtained. This means that the rate constants used in the models do not directly correspond to biological data. However the relationships between the different rate constants are representative of biological data. It is important to note that this data is very preliminary and only demonstrates that the modelling approach we have taken can be used to investigate the factors affecting protein misfolding.<br />
</p><br />
<br />
<h3>Result Case 1: Successful expression of stable protein</h3><br />
<p>In this case the initial amount of peptide produced is very stable and the equilibrium favours the fast formation of stable correctly folded protein ( red line). Unstable protein is still produced in this scenario, but is quickly degraded by and does not form inclusion bodies. Additionally the unstable protein is not exposing significant hydrophobic amino acids. This also helps prevent the formation of inclusion bodies.</p><br />
<img style="margin-left:-15px; width:642px; height:371px" src="https://static.igem.org/mediawiki/2010/b/bf/Stable_production.png" /><br /><br />
<table><br />
<p><b>Figure 3. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 M ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>There is still a peak in unstable protein, as this species will still be present. But this amount will be degraded quickly.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the natal peptide is quickly driven towards stable protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>In this scenario very few inclusion bodies are produced as there is not enough unstable protein present to induce nucleation</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h3>Result Case 2: High hydrophobic to charged amino acid ratio with a protein having many unstable intermediates </h3><br />
<p><br />
In this case the produced peptide is highly unstable and is composed of significantly more hydrophobic amino acids than charged amino acids. This indicates that in the unstable form a significant amount of the exposed amino acids will be hydrophobic. This state of unstable protein will strongly drive towards inclusion body formation.<br />
</p><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/a/a7/Highly_unstable.png" /><br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>A high hydropathy value increases the time required for the protein to fold into its correct shape. This means more unstable protein will be present in the equilibrium. The green line on the graph represents this value. The peak on the graph represents the point of nucleation whereby the concentration of unstable protein reaches a peak that rapidly increases the drive to inclusion bodies.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>The increased hydropathy content decreases the presence of functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line )</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<br />
<br />
<h3>Result Case 3: Over produced protein at high temperature</h3><br />
<p>In this scenario the protein is highly stable and drives strongly towards it’s stable functional conformation. However the over production of the peptide means there are high concentrations of unstable protein present, and the higher temperature increases the kinetic movement of the unstable protein. This causes the unstable proteins to collide more frequently and form inclusion bodies before they have a chance to become stable protein. Since the protein is highly stable the nucleation point, the peak of the green line, occurs at a higher concentration and greater time value than that of the unstable protein in the previous case.</p><br /><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/3/31/Hightemp_stable.png" /><br /> <br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>The highly stable protein produced in this scenario spends very little time in the unstable form. It is quickly converted to stable protein or driven into an inclusion body. In this scenario the concentration and temperature causes the equilibrium to favor inclusion bodies over the functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the functional protein is very stable and favored. However the concentration of unstable intermediate causes the intermediate proteins to be caught up in inclusion bodies prior to formation of correctly folded protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line ). This point is reached more slowly than in the previous scenario as the protein is more stable, and a certain concentration must be met before nucleation occurs.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h2 id="Conclusions" style="color:#0066CC">Conclusions</h2><br />
<p> <br />
There are a number of immediate problems with this model approach that affect the accuracy of the results. The first and most significant is that the values for the amounts of species and the kinetic constants were selected for convenience. This means they were selected such that their relative relationships would allow us to determine if the model could be made to match the behaviour seen in the literature. As a result the the outputs of the simulation can not be taken as "real" because they do not use true values. The second major issue is the equilibrium equations proposed for each of the reactions are abstractions and may not be the best ways of relating the species. The third major issue is that there aren't any clearly defined relationships between each of the factors being investigated and misfiling. Therefore the relationship between factors such as temperature and sequence features such as hydrophobic/charged amino acids are accounted for in a qualitative way only.<br />
</p><br />
<p>Accounting for these caveats this model was still a success in a number of ways. The results demonstrate that the MATLAB Simbiology software can be used to simulate the process of inclusion body formation. The graphs obtained match closely, albeit in a qualitative way, the process of inclusion body formation as it is described in the literature. Lastly, this approach has provided us with a framework that can be used to study the factors affecting protein misfolding and aggregation. <br />
</p><br /><br />
<br />
<h2 id="Future" style="color:#0066CC">Future directions</h2><br />
<p>The most necessary future direction is to find a "test" protein and apply the principles of the model to determine if the simulation results match with literature results. If this can be shown more test proteins can be evaluated with the model and the model can be made more general. The second future direction is to explore the concept of "cut off" values. From the equilibrium graphs in the results section it is clear that there is some amount of inclusion body present, some amount of unstable protein and some amount of stable functional protein. This implies that there will always be a ratio between the three different species and also that even when inclusion body is present some functional protein will be too. A cut off value would be the ratio of functional protein to non functional such that functional protein can still be obtained. The final future direction is to develop a way to account for the four different categories of inclusion bodies that are seen in the literature values. <br />
</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Modelling/MATLABTeam:Calgary/Modelling/MATLAB2010-10-27T21:55:11Z<p>Pauladamiak: </p>
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<h1>Modelling</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/MATLAB">MATLAB Models</a><br />
<ul><br />
<li><a href="#MATLAB">Matrix Laboratory Software</a></li><br />
<li><a href="#Protein">Protein production abstraction</a></li><br />
<li><a href="#Relationships">Relationship equations</a></li><br />
<li><a href="#Factors">Factors under investigation</a></li><br />
<li><a href="#Results">Result cases</a></li><br />
<li><a href="#Conclusions">Conclusions</a></li><br />
<li><a href="#Future">Future directions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/Maya">Maya Animations</a></li><br />
<ul><br />
<br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1 id="MATLAB">MATLAB</h1></span><br />
<h2 style="color:#0066CC">Matrix Laboratory Software </h2><br />
<p>The protein production simulation was produced using the Matrix Laboratory software, MATLAB produced by MathWorks. Specifically this project uses the Simbiology application, which is a collection of computational tools for simulating biological processes. The power of this tool lies in its ability to build multi species and reaction models, then simulate how the species will interact.<br />
</p><br /><br />
<br />
<br />
<h2 id="Protein" style="color:#0066CC">Protein production abstraction</h2><br />
<p>Our proposed model relies on an abstraction of the process of protein production, taking into account the formation of incorrectly folded intermediates and the presence of aggregated misfolded protein. Our proposed model is shown below</p> <br />
<table><br />
<tr><td valign="top"><img style="margin-right:5px;" src="https://static.igem.org/mediawiki/2010/2/24/Model_flow_chart.png" title="MATLAB model network" /></td><br />
<td align="justify"><p><b>Figure 1.</b> The proposed path consists of a number of species. The first one identified is "natal peptide" this species represents the initial amount of amino acid chain being produced by the ribosome. It is the "starting" amount of potential protein/misfolded protein/inclusion body present in the system.</p><br />
<p>The second species identified is the "unstable protein" this references proteins that have not reached their fully stable conformations yet or have been destabilized by environmental conditions. Unstable protein has the potential to be degraded by cellular proteases which results in the "degraded protein" species. Additionally unstable proteins have the potential to clump together to form the "inclusion body" species. The inclusion body species is also degraded by proteases so the degraded protein species is also a potential result.</p><br />
<p>The final species is the stable functional protein form. This stable form is also degraded by proteases but in very very small amounts</p> <br />
</td></tr><br />
</table><br /><br />
<br />
<br />
<h2 id="Relationships" style="color:#0066CC">Relationship equations</h2><br />
<p>In order to begin investigating the factors that may affect the final state of the natal peptide it first becomes important to define how each of the species in the system interact. This is where the MATLAB software becomes useful. The proposed model pathway can be represented in the MATLAB Simbiology Toolbox.</p><br />
<table><br />
<tr><br />
<td valign="top" align="justify"><b>Figure 2.</b> Each of the blue circles correspond to the species identified in the previous section, while the yellow circles define the reaction that occurs between the two species. The Simbiology software uses these defined reactions to determine the amounts of each species present in the system as the species interact over a period of time. The results of the simulation are described in a later section<br />
</td><br />
<td><br />
<img style="margin-left:5px; width:409px; height:347px;" src="https://static.igem.org/mediawiki/2010/1/12/Matlab_network.png" title="MATLAB Diagram View" /><br />
</td><br />
</tr><br />
</table><br /><br />
<p>The reactions present in the system are described by the following equations</p><br /><br />
<table><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>natal peptide -> unstable protein</em></td><br />
<td><p>This reaction is an irreversible reaction where all of the natal peptide present becomes unstable protein with a rate constant of 1. This reflects the assumption that all of the initial amino acid sequence will at some point be present as unstable protein capable of being degraded or forming inclusion bodies</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein + inclusion body <-> 2 inclusion body</em></td><br />
<td><p>This reaction is a reversible reaction that defines how unstable proteins form into inclusion bodies. The simulation assumes that there is a baseline very small concentration of inclusion body present. This base value may interact with unstable protein to form more inclusion bodies resulting in a positive feed back that begins to form higher concentrations of inclusion bodies. The rate constant at which this process occurs is dependent on a number of factors discussed in a later section. Inclusion body formation represents a potential outcome of the protein expression system</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>unstable protein -> degraded protein</em></td><br />
<td><p>This reaction represents unstable protein being degraded by proteases. This reaction follows the Henri-Michaelis-Menten equilibrium process. In this process there is a pseudo equilibrium present that is determined to be one way, as degraded protein does not return to its pre degraded state. The constant for this reaction is a set value that is relative to the particular protease. For simplicity a single value was selected to represent this impact. Once the unstable protein has been degraded it is removed from the simulation system and can not form inclusion bodies or functional proteins. As a result degraded protein represents one of the potential "outcomes" of a protein expression experiment</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>inclusion body -> degraded protein</em></td><br />
<td><p>Our system assumes that inclusion bodies are degraded by the same process as unstable proteins, but that the rate of degradation is slower than that of a single unstable protein. This is proposed to be the result of the protease being unable to access individual proteins of the inclusion body for degradation. As a result the process is considerably slower than the degradation of straight unstable proteins. The difference is reflected in the rate constant for each of the two reactions</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein <-> functional protein</em></td><br />
<td><p>This reaction represents the process of the unstable protein becoming stable functional protein. The reaction is reversible as different environmental conditions, discussed later on, can determine whether or not the protein is in a stable state or unstable state.</p><p></p></td><br />
</tr></table><br />
<br /><br />
<br />
<br />
<h2 id="Factors" style="color:#0066CC">Factors under investigation</h2><br />
<p> For the purpose of our model there were two categories of factors that we investigated. These categories are Environmental factors and sequence factors. It is important to note that these divisions are arbitrary and don't necessarily exist in reality. Specifically these categories were created for the convenience of organizing the factors being investigated and determining how to best evaluate their impact.</p><br />
<h3>Environmental factors</h3><br />
<p>The collection of factors refers to those features of the cells environment that will have an impact on the stability of proteins being produced. When we use the term environment we are referring to both the external temperature at which the cell is growing, the pH of its environment and the concentration of protein within cellular compartments. These factors are not determined by features of the protein being produced but will still affect the likelihood of inclusion body formation and/or protein instability.<br />
</p><br />
<h4 style="color:#003366">Defining environmental impact on protein stability</h4><br />
<p>We have assumed that environmental conditions affect inclusion body formation by altering the equilibrium of the following equation:</p><br />
<br />
<div style="width:450px; margin-left:auto; margin-right:auto;"><b>Functional Protein <-> Unstable Protein <-> Inclusion Body </b></div><br /><br />
<br />
<p>A publication by Brandt et al supports this concept as they showed that isolated protein in a solution can be converted back and forth from its stable form and inclusion body form based on temperature and pH. Specifically that high temperatures and strongly basic pH will encourage the formation of inclusion bodies. This means that equilibrium constant increases as the formation of inclusion bodies has become more favourable</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>From this information we have assumed that altering the environmental conditions of temperature, pH and protein concentration will have a quantifiable effect on the rate constants for the above equation. Currently the exact impact of these factors hasn't been determined. The results section only describes rate constants determined for convenience based on qualitative understanding of the processes.<br />
</p><br />
<br />
<h3>Sequence dependent factors</h3> <br />
<p>For these factors we have tried to look at the features of the mRNA and amino acid sequence that could impact the likelihood of inclusion body formation. As a general rule sequence factors were selected based on how the particular feature affects the time taken for the sequence to reach its fully folded stable confirmation. The reason for this based on the assumption that if the protein has more intermediate stages or is more thermodynamically stable in a non folded confirmation then there is more time for the unstable proteins to interact and begin the formation of inclusion bodies.</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>Sequence features such as scarce amino acids ( Tryptohphan ), mRNA structural features that inhibit translation time through the ribosome and the ratio of hydrophobic amino acids to charged amino acids all have a quantifiable effect on the rate constant at which unstable protein becomes stable protein.</p><br />
<br />
<p>This evidence has been indirectly supported from the literature and discussions with researchers in the field. However at this point the precise impact of these factors is still under investigation. As such for the results section, hypothesized relationships alone have been used.</p><br /><br />
<br />
<br />
<br />
<br />
<h2 id="Results" style="color:#0066CC">Result cases</h2><br />
<p>The result cases represent the preliminary testing of the model to see if the model simulation can be used to analyze the protein production process under different conditions. The rate constants used for each of the cases were selected for convenience in order to determine if relevant results could be obtained. This means that the rate constants used in the models do not directly correspond to biological data. However the relationships between the different rate constants are representative of biological data. It is important to note that this data is very preliminary and only demonstrates that the modelling approach we have taken can be used to investigate the factors affecting protein misfolding.<br />
</p><br />
<br />
<h3>Result Case 1: Successful expression of stable protein</h3><br />
<p>In this case the initial amount of peptide produced is very stable and the equilibrium favours the fast formation of stable correctly folded protein ( red line). Unstable protein is still produced in this scenario, but is quickly degraded by and does not form inclusion bodies. Additionally the unstable protein is not exposing significant hydrophobic amino acids. This also helps prevent the formation of inclusion bodies.</p><br />
<img style="margin-left:-15px; width:722px; height:417px" src="https://static.igem.org/mediawiki/2010/b/bf/Stable_production.png" /><br /><br />
<table><br />
<p><b>Figure 3. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 M ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>There is still a peak in unstable protein, as this species will still be present. But this amount will be degraded quickly.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the natal peptide is quickly driven towards stable protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>In this scenario very few inclusion bodies are produced as there is not enough unstable protein present to induce nucleation</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h3>Result Case 2: High hydrophobic to charged amino acid ratio with a protein having many unstable intermediates </h3><br />
<p><br />
In this case the produced peptide is highly unstable and is composed of significantly more hydrophobic amino acids than charged amino acids. This indicates that in the unstable form a significant amount of the exposed amino acids will be hydrophobic. This state of unstable protein will strongly drive towards inclusion body formation.<br />
</p><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/a/a7/Highly_unstable.png" /><br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>A high hydropathy value increases the time required for the protein to fold into its correct shape. This means more unstable protein will be present in the equilibrium. The green line on the graph represents this value. The peak on the graph represents the point of nucleation whereby the concentration of unstable protein reaches a peak that rapidly increases the drive to inclusion bodies.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>The increased hydropathy content decreases the presence of functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line )</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<br />
<br />
<h3>Result Case 3: Over produced protein at high temperature</h3><br />
<p>In this scenario the protein is highly stable and drives strongly towards it’s stable functional conformation. However the over production of the peptide means there are high concentrations of unstable protein present, and the higher temperature increases the kinetic movement of the unstable protein. This causes the unstable proteins to collide more frequently and form inclusion bodies before they have a chance to become stable protein. Since the protein is highly stable the nucleation point, the peak of the green line, occurs at a higher concentration and greater time value than that of the unstable protein in the previous case.</p><br /><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/3/31/Hightemp_stable.png" /><br /> <br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>The highly stable protein produced in this scenario spends very little time in the unstable form. It is quickly converted to stable protein or driven into an inclusion body. In this scenario the concentration and temperature causes the equilibrium to favor inclusion bodies over the functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the functional protein is very stable and favored. However the concentration of unstable intermediate causes the intermediate proteins to be caught up in inclusion bodies prior to formation of correctly folded protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line ). This point is reached more slowly than in the previous scenario as the protein is more stable, and a certain concentration must be met before nucleation occurs.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h2 id="Conclusions" style="color:#0066CC">Conclusions</h2><br />
<p> <br />
There are a number of immediate problems with this model approach that affect the accuracy of the results. The first and most significant is that the values for the amounts of species and the kinetic constants were selected for convenience. This means they were selected such that their relative relationships would allow us to determine if the model could be made to match the behaviour seen in the literature. As a result the the outputs of the simulation can not be taken as "real" because they do not use true values. The second major issue is the equilibrium equations proposed for each of the reactions are abstractions and may not be the best ways of relating the species. The third major issue is that there aren't any clearly defined relationships between each of the factors being investigated and misfiling. Therefore the relationship between factors such as temperature and sequence features such as hydrophobic/charged amino acids are accounted for in a qualitative way only.<br />
</p><br />
<p>Accounting for these caveats this model was still a success in a number of ways. The results demonstrate that the MATLAB Simbiology software can be used to simulate the process of inclusion body formation. The graphs obtained match closely, albeit in a qualitative way, the process of inclusion body formation as it is described in the literature. Lastly, this approach has provided us with a framework that can be used to study the factors affecting protein misfolding and aggregation. <br />
</p><br /><br />
<br />
<h2 id="Future" style="color:#0066CC">Future directions</h2><br />
<p>The most necessary future direction is to find a "test" protein and apply the principles of the model to determine if the simulation results match with literature results. If this can be shown more test proteins can be evaluated with the model and the model can be made more general. The second future direction is to explore the concept of "cut off" values. From the equilibrium graphs in the results section it is clear that there is some amount of inclusion body present, some amount of unstable protein and some amount of stable functional protein. This implies that there will always be a ratio between the three different species and also that even when inclusion body is present some functional protein will be too. A cut off value would be the ratio of functional protein to non functional such that functional protein can still be obtained. The final future direction is to develop a way to account for the four different categories of inclusion bodies that are seen in the literature values. <br />
</p><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Modelling/MATLABTeam:Calgary/Modelling/MATLAB2010-10-27T21:53:22Z<p>Pauladamiak: </p>
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<h1>Modelling</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/MATLAB">MATLAB Models</a><br />
<ul><br />
<li><a href="#MATLAB">Matrix Laboratory Software</a></li><br />
<li><a href="#Protein">Protein production abstraction</a></li><br />
<li><a href="#Relationships">Relationship equations</a></li><br />
<li><a href="#Factors">Factors under investigation</a></li><br />
<li><a href="#Results">Result cases</a></li><br />
<li><a href="#Conclusions">Conclusions</a></li><br />
<li><a href="#Future">Future directions</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Modelling/Maya">Maya Animations</a></li><br />
<ul><br />
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<span id="bodytitle"><h1 id="MATLAB">MATLAB</h1></span><br />
<h2 style="color:#0066CC">Matrix Laboratory Software </h2><br />
<p>The protein production simulation was produced using the Matrix Laboratory software, MATLAB produced by MathWorks. Specifically this project uses the Simbiology application, which is a collection of computational tools for simulating biological processes. The power of this tool lies in its ability to build multi species and reaction models, then simulate how the species will interact.<br />
</p><br /><br />
<br />
<br />
<h2 id="Protein" style="color:#0066CC">Protein production abstraction</h2><br />
<p>Our proposed model relies on an abstraction of the process of protein production, taking into account the formation of incorrectly folded intermediates and the presence of aggregated misfolded protein. Our proposed model is shown below</p> <br />
<table><br />
<tr><td valign="top"><img style="margin-right:5px;" src="https://static.igem.org/mediawiki/2010/2/24/Model_flow_chart.png" title="MATLAB model network" /></td><br />
<td align="justify"><p><b>Figure 1.</b> The proposed path consists of a number of species. The first one identified is "natal peptide" this species represents the initial amount of amino acid chain being produced by the ribosome. It is the "starting" amount of potential protein/misfolded protein/inclusion body present in the system.</p><br />
<p>The second species identified is the "unstable protein" this references proteins that have not reached their fully stable conformations yet or have been destabilized by environmental conditions. Unstable protein has the potential to be degraded by cellular proteases which results in the "degraded protein" species. Additionally unstable proteins have the potential to clump together to form the "inclusion body" species. The inclusion body species is also degraded by proteases so the degraded protein species is also a potential result.</p><br />
<p>The final species is the stable functional protein form. This stable form is also degraded by proteases but in very very small amounts</p> <br />
</td></tr><br />
</table><br /><br />
<br />
<br />
<h2 id="Relationships" style="color:#0066CC">Relationship equations</h2><br />
<p>In order to begin investigating the factors that may affect the final state of the natal peptide it first becomes important to define how each of the species in the system interact. This is where the MATLAB software becomes useful. The proposed model pathway can be represented in the MATLAB Simbiology Toolbox.</p><br />
<table><br />
<tr><br />
<td valign="top" align="justify"><b>Figure 2.</b> Each of the blue circles correspond to the species identified in the previous section, while the yellow circles define the reaction that occurs between the two species. The Simbiology software uses these defined reactions to determine the amounts of each species present in the system as the species interact over a period of time. The results of the simulation are described in a later section<br />
</td><br />
<td><br />
<img style="margin-left:5px; width:409px; height:347px;" src="https://static.igem.org/mediawiki/2010/1/12/Matlab_network.png" title="MATLAB Diagram View" /><br />
</td><br />
</tr><br />
</table><br /><br />
<p>The reactions present in the system are described by the following equations</p><br /><br />
<table><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>natal peptide -> unstable protein</em></td><br />
<td><p>This reaction is an irreversible reaction where all of the natal peptide present becomes unstable protein with a rate constant of 1. This reflects the assumption that all of the initial amino acid sequence will at some point be present as unstable protein capable of being degraded or forming inclusion bodies</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein + inclusion body <-> 2 inclusion body</em></td><br />
<td><p>This reaction is a reversible reaction that defines how unstable proteins form into inclusion bodies. The simulation assumes that there is a baseline very small concentration of inclusion body present. This base value may interact with unstable protein to form more inclusion bodies resulting in a positive feed back that begins to form higher concentrations of inclusion bodies. The rate constant at which this process occurs is dependent on a number of factors discussed in a later section. Inclusion body formation represents a potential outcome of the protein expression system</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>unstable protein -> degraded protein</em></td><br />
<td><p>This reaction represents unstable protein being degraded by proteases. This reaction follows the Henri-Michaelis-Menten equilibrium process. In this process there is a pseudo equilibrium present that is determined to be one way, as degraded protein does not return to its pre degraded state. The constant for this reaction is a set value that is relative to the particular protease. For simplicity a single value was selected to represent this impact. Once the unstable protein has been degraded it is removed from the simulation system and can not form inclusion bodies or functional proteins. As a result degraded protein represents one of the potential "outcomes" of a protein expression experiment</p><p></p></td></tr><br />
<tr><td style="width:175px; margin-right:5px;" valign="top"><em>inclusion body -> degraded protein</em></td><br />
<td><p>Our system assumes that inclusion bodies are degraded by the same process as unstable proteins, but that the rate of degradation is slower than that of a single unstable protein. This is proposed to be the result of the protease being unable to access individual proteins of the inclusion body for degradation. As a result the process is considerably slower than the degradation of straight unstable proteins. The difference is reflected in the rate constant for each of the two reactions</p><p></p></td></tr><br />
<tr><td style="width:250px; margin-right:5px;" valign="top"><em>unstable protein <-> functional protein</em></td><br />
<td><p>This reaction represents the process of the unstable protein becoming stable functional protein. The reaction is reversible as different environmental conditions, discussed later on, can determine whether or not the protein is in a stable state or unstable state.</p><p></p></td><br />
</tr></table><br />
<br /><br />
<br />
<br />
<h2 id="Factors" style="color:#0066CC">Factors under investigation</h2><br />
<p> For the purpose of our model there were two categories of factors that we investigated. These categories are Environmental factors and sequence factors. It is important to note that these divisions are arbitrary and don't necessarily exist in reality. Specifically these categories were created for the convenience of organizing the factors being investigated and determining how to best evaluate their impact.</p><br />
<h3>Environmental factors</h3><br />
<p>The collection of factors refers to those features of the cells environment that will have an impact on the stability of proteins being produced. When we use the term environment we are referring to both the external temperature at which the cell is growing, the pH of its environment and the concentration of protein within cellular compartments. These factors are not determined by features of the protein being produced but will still affect the likelihood of inclusion body formation and/or protein instability.<br />
</p><br />
<h4 style="color:#003366">Defining environmental impact on protein stability</h4><br />
<p>We have assumed that environmental conditions affect inclusion body formation by altering the equilibrium of the following equation:</p><br />
<br />
<div style="width:450px; margin-left:auto; margin-right:auto;"><b>Functional Protein <-> Unstable Protein <-> Inclusion Body </b></div><br /><br />
<br />
<p>A publication by Brandt et al supports this concept as they showed that isolated protein in a solution can be converted back and forth from its stable form and inclusion body form based on temperature and pH. Specifically that high temperatures and strongly basic pH will encourage the formation of inclusion bodies. This means that equilibrium constant increases as the formation of inclusion bodies has become more favourable</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>From this information we have assumed that altering the environmental conditions of temperature, pH and protein concentration will have a quantifiable effect on the rate constants for the above equation. Currently the exact impact of these factors hasn't been determined. The results section only describes rate constants determined for convenience based on qualitative understanding of the processes.<br />
</p><br />
<br />
<h3>Sequence dependent factors</h3> <br />
<p>For these factors we have tried to look at the features of the mRNA and amino acid sequence that could impact the likelihood of inclusion body formation. As a general rule sequence factors were selected based on how the particular feature affects the time taken for the sequence to reach its fully folded stable confirmation. The reason for this based on the assumption that if the protein has more intermediate stages or is more thermodynamically stable in a non folded confirmation then there is more time for the unstable proteins to interact and begin the formation of inclusion bodies.</p><br />
<br />
<h4 style="color:#003366">Critical assumption</h4><br />
<p>Sequence features such as scarce amino acids ( Tryptohphan ), mRNA structural features that inhibit translation time through the ribosome and the ratio of hydrophobic amino acids to charged amino acids all have a quantifiable effect on the rate constant at which unstable protein becomes stable protein.</p><br />
<br />
<p>This evidence has been indirectly supported from the literature and discussions with researchers in the field. However at this point the precise impact of these factors is still under investigation. As such for the results section, hypothesized relationships alone have been used.</p><br /><br />
<br />
<br />
<br />
<br />
<h2 id="Results" style="color:#0066CC">Result cases</h2><br />
<p>The result cases represent the preliminary testing of the model to see if the model simulation can be used to analyze the protein production process under different conditions. The rate constants used for each of the cases were selected for convenience in order to determine if relevant results could be obtained. This means that the rate constants used in the models do not directly correspond to biological data. However the relationships between the different rate constants are representative of biological data. It is important to note that this data is very preliminary and only demonstrates that the modelling approach we have taken can be used to investigate the factors affecting protein misfolding.<br />
</p><br />
<br />
<h3>Result Case 1: Successful expression of stable protein</h3><br />
<p>In this case the initial amount of peptide produced is very stable and the equilibrium favours the fast formation of stable correctly folded protein ( red line). Unstable protein is still produced in this scenario, but is quickly degraded by and does not form inclusion bodies. Additionally the unstable protein is not exposing significant hydrophobic amino acids. This also helps prevent the formation of inclusion bodies.</p><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/b/bf/Stable_production.png" /><br /><br />
<table><br />
<p><b>Figure 3. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 M ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>There is still a peak in unstable protein, as this species will still be present. But this amount will be degraded quickly.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the natal peptide is quickly driven towards stable protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>In this scenario very few inclusion bodies are produced as there is not enough unstable protein present to induce nucleation</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h3>Result Case 2: High hydrophobic to charged amino acid ratio with a protein having many unstable intermediates </h3><br />
<p><br />
In this case the produced peptide is highly unstable and is composed of significantly more hydrophobic amino acids than charged amino acids. This indicates that in the unstable form a significant amount of the exposed amino acids will be hydrophobic. This state of unstable protein will strongly drive towards inclusion body formation.<br />
</p><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/a/a7/Highly_unstable.png" /><br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>A high hydropathy value increases the time required for the protein to fold into its correct shape. This means more unstable protein will be present in the equilibrium. The green line on the graph represents this value. The peak on the graph represents the point of nucleation whereby the concentration of unstable protein reaches a peak that rapidly increases the drive to inclusion bodies.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>The increased hydropathy content decreases the presence of functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line )</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<br />
<br />
<h3>Result Case 3: Over produced protein at high temperature</h3><br />
<p>In this scenario the protein is highly stable and drives strongly towards it’s stable functional conformation. However the over production of the peptide means there are high concentrations of unstable protein present, and the higher temperature increases the kinetic movement of the unstable protein. This causes the unstable proteins to collide more frequently and form inclusion bodies before they have a chance to become stable protein. Since the protein is highly stable the nucleation point, the peak of the green line, occurs at a higher concentration and greater time value than that of the unstable protein in the previous case.</p><br /><br />
<img style="margin-left:-15px; width:803px; height:464px" src="https://static.igem.org/mediawiki/2010/3/31/Hightemp_stable.png" /><br /> <br />
<p><b>Figure 5. </b>In this figure the concentration units are arbitrarily mM and the time value is in seconds. These units were selected for convenience and are not assumed to be accurate for the process being modelled. This issue is discussed in more detail in the conclusion section</p><br />
<table><br />
<tr><br />
<td valign="top" style="width:175px"><p><em>Natal peptide(blue line)</em></p><p></p></td><br />
<td valign="top"><p>An initial arbitrary amount of natal peptide ( 10 mM ) is produced in the cell</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Unstable protein (green line)</em><p></p></td><br />
<td valign="top"><p>The highly stable protein produced in this scenario spends very little time in the unstable form. It is quickly converted to stable protein or driven into an inclusion body. In this scenario the concentration and temperature causes the equilibrium to favor inclusion bodies over the functional protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Functional protein (red line)</em><p></p></td><br />
<td valign="top"><p>In this scenario the functional protein is very stable and favored. However the concentration of unstable intermediate causes the intermediate proteins to be caught up in inclusion bodies prior to formation of correctly folded protein.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><p><em>Inclusion body (light blue line) </em></p><p></p></td><br />
<td valign="top"><p>The concentration of inclusion body increases rapidly when the concentration of unstable protein reaches the point of nucleation ( peak of the green line ). This point is reached more slowly than in the previous scenario as the protein is more stable, and a certain concentration must be met before nucleation occurs.</p><p></p></td><br />
</tr><br />
<br />
<tr><br />
<td valign="top"><em>Degraded protein (purple line)</em><p></p></td><br />
<td valign="top"><p>Unstable protein and inclusion bodies are degraded. In this model unstable protein is degraded faster than inclusion bodies due to the size difference</p><p></p></td><br />
</tr><br />
</table><br />
<br /><br />
<br />
<h2 id="Conclusions" style="color:#0066CC">Conclusions</h2><br />
<p> <br />
There are a number of immediate problems with this model approach that affect the accuracy of the results. The first and most significant is that the values for the amounts of species and the kinetic constants were selected for convenience. This means they were selected such that their relative relationships would allow us to determine if the model could be made to match the behaviour seen in the literature. As a result the the outputs of the simulation can not be taken as "real" because they do not use true values. The second major issue is the equilibrium equations proposed for each of the reactions are abstractions and may not be the best ways of relating the species. The third major issue is that there aren't any clearly defined relationships between each of the factors being investigated and misfiling. Therefore the relationship between factors such as temperature and sequence features such as hydrophobic/charged amino acids are accounted for in a qualitative way only.<br />
</p><br />
<p>Accounting for these caveats this model was still a success in a number of ways. The results demonstrate that the MATLAB Simbiology software can be used to simulate the process of inclusion body formation. The graphs obtained match closely, albeit in a qualitative way, the process of inclusion body formation as it is described in the literature. Lastly, this approach has provided us with a framework that can be used to study the factors affecting protein misfolding and aggregation. <br />
</p><br /><br />
<br />
<h2 id="Future" style="color:#0066CC">Future directions</h2><br />
<p>The most necessary future direction is to find a "test" protein and apply the principles of the model to determine if the simulation results match with literature results. If this can be shown more test proteins can be evaluated with the model and the model can be made more general. The second future direction is to explore the concept of "cut off" values. From the equilibrium graphs in the results section it is clear that there is some amount of inclusion body present, some amount of unstable protein and some amount of stable functional protein. This implies that there will always be a ratio between the three different species and also that even when inclusion body is present some functional protein will be too. A cut off value would be the ratio of functional protein to non functional such that functional protein can still be obtained. The final future direction is to develop a way to account for the four different categories of inclusion bodies that are seen in the literature values. <br />
</p><br />
</div><br />
<br />
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<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/File:Highly_unstable.pngFile:Highly unstable.png2010-10-27T21:25:38Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:Hightemp_stable.pngFile:Hightemp stable.png2010-10-27T21:09:13Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:StableProteinProduction.pngFile:StableProteinProduction.png2010-10-27T20:30:10Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:Stable_production.pngFile:Stable production.png2010-10-27T20:25:20Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:Matlab_network.pngFile:Matlab network.png2010-10-27T19:34:24Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:Model_flow_chart.pngFile:Model flow chart.png2010-10-27T19:27:01Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Project/ControlsTeam:Calgary/Project/Controls2010-10-27T16:09:13Z<p>Pauladamiak: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing parameters</a></li><br />
<br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Testing our system</h1></span><br />
<br />
<p><br />
Once constructed, we needed a way to test the cytoplasm and periplasmic stress promoters in order to characterize them. We did this in three different ways.<br />
</p><br />
<ol><br />
<li>Testing with known folding and misfolding proteins<br /></li><br />
<li>Testing with NlpE, an outer membrane lipoprotein known to activate the Cpx regulon<br /></li><br />
<li>Testing with varying temperature conditions<br /></li><br />
</ol><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with known folding and misfolding proteins</h2><br />
<p><br />
First, it was necessary to identify proteins that are known successful folders and known non-folders in <i> E. coli</i>. Maltose binding protein was selected for this purpose. Wild type maltose binding protein (MalE) is transporter protein that is shuttled to the periplasmic space of <i>E.coli</i> and known to fold extremely well there. We chose a mutant form of maltose binding protein, MalE31, that does not fold in the periplasm due to two amino acid substitutions in positions 33 and 34. In addition to this periplasmic mutant, another mutant form of MalE was found, with the signal sequence required for transport to the periplasmic space deleted. The MalEΔSS folds extremely well in the cytoplasm regardless of the deletion. MalE31 with its signal sequence removed is a non folder in the cytoplasm. Thus, we have four proteins covering folding and non-folding in both the periplasm and the cytoplasm.<br />
</p><br />
<div style="width:400px; height:400px; border:1px solid black"><p>maltose binding chart place holder</div><br /><br />
<br />
<p>The wild type MalE as well as the mutant versions were received from the Betton lab in France. These parts were Biobricked, but prior to testing the selected stress reporter circuits with these parts, it was necessary to test these MalE variations to ensure that they were functional and matched literature data. To do this, we transformed them into strains of cells containing cpxR and degP promoters upstream of a lacZ rpeorter (Raivio labs). We would expect malE31, if it misfolded, to activate the cpxR and degP stress promoters, thus providing a blue output from lacZ. MalE, on the other hand, is expected to fold properly, thus not activating these promoters, and produce any lacZ activity. These assays allowed us to conclude that MalE and MalE31 work the way that we expected them to. See results on our characterization page.</p><br />
<br />
<p><br />
Once malE and malE31 were shown to be functional, we then used them to test out the stress promoters. We did this by making competent cells containing our reporter circuits. We then transformed in inducible constructs containing our MalE and mutant MalE proteins. Fluorescent output was measured from these assays and the results can be seen on our characterization page.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with NLPE</h2><br />
<p><br />
NLPE is an outer membrane lipoprotein that literature has shown to activate the Cpx pathway. We transformed expression constructs for this protein (obtained from Dr. Tracy Raivio's lab) into Top10 competent cells containing our CpxR reporter and looked for fluorescent output. Results for this experiment can be viewed on our characterization page.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with Varying Temperature Conditions</h2><br />
<p><br />
Finally we tested the CpxR promoter activity in the presence of varying temperature conditions. Literature data has shown that the Cpx pathway is also activated due to heat stresses. To establish a baseline that could be used to determine whether heat misfolded protein was activating the pathway, we exposed cells containing the CpxR promoter to heat stresses of 30&deg;C, 37 &deg;C, 42 &deg;C, and 47 &deg;C. The results can be seen on our characterization page under Experiment 2.<br />
</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/SafetyTeam:Calgary/Notebook/Safety2010-10-27T10:37:47Z<p>Pauladamiak: </p>
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<span id="bodytitle"><h1>iGEM Safety Questionnaire</h1></span><br />
<p><br />
As a safety precaution, all Igem team members have been fully trained in WHIMIS as well as in introductory Biosfatey.<br />
</p><br /><br />
<p><br />
<em>Would any of your project ideas raise safety issues in terms of:</em><br /> <br />
<em>researcher safety</em><br />
<em>public safety, or</em> <br />
<em>environmental safety?</em> <br />
</p><br />
<p><br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the context of the iGEM competiion and beyond. For this reason, our project only really poses safety issues if it was to be used to aid in the expression of toxic or otherwise dangerous proteins.<br />
</p><br />
<br /><br />
<p><em>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</em></p> <br />
<p><br />
No, none of the parts that we made alone raise any safety issues. Again, our project only has the possibility of posing safety issues in regards to the final product the user of our tool kit is trying to produce.<br />
</p><br />
<br /><br />
<p><br />
<em>Is there a local biosafety group, committee, or review board at your institution?</em><br />
</p> <br />
<p><br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.</p><br />
<br /><br />
<p><br />
<em>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</em></p><br />
<br />
<p><br />
insert answer here!<br />
</p><br />
<br /><br />
<br />
<br />
<br />
. <br />
<br />
<br />
<br />
<br />
</div><br />
<br />
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<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/SafetyTeam:Calgary/Notebook/Safety2010-10-27T10:37:24Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">Safety</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<span id="bodytitle"><h1>iGEM Safety Questionnaire</h1></span><br />
<p><br />
As a safety precaution, all Igem team members have been fully trained in WHIMIS as well as in introductory Biosfatey.<br />
</p><br />
<p><br />
<em>Would any of your project ideas raise safety issues in terms of:</em><br /> <br />
<em>researcher safety</em><br />
<em>public safety, or</em> <br />
<em>environmental safety?</em> <br />
</p><br />
<p><br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the context of the iGEM competiion and beyond. For this reason, our project only really poses safety issues if it was to be used to aid in the expression of toxic or otherwise dangerous proteins.<br />
</p><br />
<br /><br />
<p><em>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</em></p> <br />
<p><br />
No, none of the parts that we made alone raise any safety issues. Again, our project only has the possibility of posing safety issues in regards to the final product the user of our tool kit is trying to produce.<br />
</p><br />
<br /><br />
<p><br />
<em>Is there a local biosafety group, committee, or review board at your institution?</em><br />
</p> <br />
<p><br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.</p><br />
<br /><br />
<p><br />
<em>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</em></p><br />
<br />
<p><br />
insert answer here!<br />
</p><br />
<br /><br />
<br />
<br />
<br />
. <br />
<br />
<br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/SafetyTeam:Calgary/Notebook/Safety2010-10-27T10:36:25Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">Safety</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<span id="bodytitle"><h1>iGEM Safety Questionnaire</h1></span><br />
<p><br />
As a safety precaution, all Igem team members have been fully trained in WHIMIS as well as in introductory Biosfatey.<br />
</p><br />
<p><br />
Would any of your project ideas raise safety issues in terms of:<br /> <br />
<em>researcher safety</em><br />
<em>public safety, or</em> <br />
<em>environmental safety?</em> <br />
</p><br />
<p><br />
No, not directly. The goal of our project is to create a tool that can help solve protein expression problems in future projects both within the context of the iGEM competiion and beyond. For this reason, our project only really poses safety issues if it was to be used to aid in the expression of toxic or otherwise dangerous proteins.<br />
</p><br />
<br />
<p><em>Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</em></p> <br />
<p><br />
No, none of the parts that we made alone raise any safety issues. Again, our project only has the possibility of posing safety issues in regards to the final product the user of our tool kit is trying to produce.<br />
</p><br />
<br />
<p><br />
<em>Is there a local biosafety group, committee, or review board at your institution?</em><br />
</p> <br />
<p><br />
Yes, we have an office of medical bioethics at our Univeristy. Our project has never come up with this office. Due to the fact that we are using non-pathogenic bacteria and that our project poses no direct safety issues to researchers the public or the environment, they have no concerns with our project at present.</p><br />
<br />
<p><br />
<em>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</em></p><br />
<br />
<p><br />
insert answer here!<br />
</p><br />
<br />
<br />
<br />
<br />
. <br />
<br />
<br />
<br />
<br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/Safety_And_ProtocolsTeam:Calgary/Notebook/Safety And Protocols2010-10-27T10:30:34Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#PCR">Taq Polymerase Chain Reaction</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#TCCIP">Transformable Competent Cell Induction Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#BTP">Bacterial Transformation Protocol </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#RD">Restriction Digest</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#Ligation">Ligation</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#MPP">Miniprep Plasmid Preparation (GenElute</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#AGE">Agarose Gel Electrophoresis </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#MOAG">Making of Agarose Gel </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#RORD">Rehydration of Registry DNA</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#CT">Construction Technique</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#OC">Overnight Cultures</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#GSP">Glycerol Stock Preparation </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#LAPPP">LB Agar Plate Preparation Protocol </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#QSMK">QIAprep Spin Miniprep Kit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#GE">Gel Extraction</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#PP">PCR Purification (Vacuum)</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">Safety</a></li><br />
</ul><br />
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<span id="bodytitle"><h1>iGEM Laboratory Procedures</h1></span><br />
<br />
<a name="PCR"></a><br />
<h2 style="color:#0066CC">Taq polymerase chain reaction</h2><br />
<br />
<i> <br /> <br /> Polymerase Chain Reaction Master Mix </i><br />
<br />
<br />
<table border="2"><br />
<tr><br />
<td> <b> Reagent </b> </td><br />
<td> <b> 1x Mix (&micro;L) </b> </td><br />
<td> <b> 5x Mix (&micro;L) </b> </td><br />
</tr><br />
<br />
<tr><br />
<td> H<sub>2</sub>O </td><br />
<td> 28 </td><br />
<td> 140 </td><br />
</tr><br />
<br />
<tr><br />
<td> 10x Buffer </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> 2 mM dNTP </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> Forward Primer (2 mM) </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> Reverse Primer (2 mM) </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> 50 mM MgCl<sub>2</sub> </td><br />
<td> 1.5 </td><br />
<td> 7.5 </td><br />
</tr><br />
<br />
<tr><br />
<td> Taq Polymerase </td><br />
<td> 0.5 </td><br />
<td> 2.5 </td><br />
</tr><br />
<br />
<tr><br />
<td> <b> Total </b> </td><br />
<td> <b> 50 </b> </td><br />
<td> <b> 250 </b> </td> <br />
</tr><br />
</table><br />
<br />
<br /><br />
<br />
<p>DNA template is required. The quantity of water decreases in proportion to the volume of DNA template added so that the total volume remains the same.<p><br />
<br />
<br /> <i> Thermocycler PCR Program </i><br />
<br />
<table border="2"><br />
<tr><br />
<td> <b> Temperature (&deg;C)</b> </td><br />
<td> <b> Time (mins) </b> </td><br />
</tr><br />
<br />
<tr><br />
<td> 95.0 </td><br />
<td> 5:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 95.0 </td><br />
<td> 1:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 55.0* </td><br />
<td> 0:30 </td><br />
</tr><br />
<br />
<tr><br />
<td> 72.0 </td><br />
<td> 1:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 72.0 </td><br />
<td> 10:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 4.0 </td><br />
<td> &infin; </td><br />
</tr><br />
<br />
</table> <br />
<br />
<br /><br />
<p> <b> NOTE: </b> * indicates that the temperature of the step is primer specific.<br />
Steps 2 through 4 are repeated 30 times.<br />
The length of Step 4 is 1 minute for every 1000 base pairs of the template to be amplified.</p><br />
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<br />
<br />
<a name="TCCIP"></a><br />
<br /> <br /> <h2 style="color:#0066CC">Making competent cells</h2><br />
<br />
<br /> <p>This procedure was done using Top10 Competent cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.</p><br />
<br />
<ol><br />
<li> Innoculate 5-10 mL LB at 37&deg;C while shaking </li><br />
<li> Subculture 1 mL of bacteria solution into 50 mL LB broth at 37&deg; while shaking<br />
until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours) </li><br />
<li> Centrifuge the subculture at 10 000 rpm at 4&deg;C for 2 minutes </li><br />
<li> Resuspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li><br />
<li> Centrifuge at 10 000 rpm at 4&deg;C for 2 minutes and resuspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li><br />
<li> Leave on ice for at least 30 minutes and then aliquot 200 uL and freeze at -80&deg;C </li><br />
</ol><br />
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<br />
<a name="BTP"></a><br />
<br /> <br /> <br />
<br />
<h2 style="color:#0066CC">Bacterial transformation</h2><br />
<br />
<br /> <br />
<ol> <br />
<li> Thaw Competent Cells </li> <br />
<li> Add 10-100 ng of DNA </li><br />
<li> Ice solution for 30 minutes </li><br />
<li> Heat shock solution (5 minutes at 37&deg;C or 2 minutes at 42&deg;C) </li><br />
<li> Ice solution for 5 minutes </li><br />
<li> Recover with 250 &micro;L of SOC (30 minutes for Ampicilin resistant plasmids and 60 minutes for kanamycin resistant plasmids) </li><br />
<li> Centrifuge for 5 seconds at 14 000 rpm and concentrate solution to 100 μL </li><br />
<li> Plate 20-50 &micro;L onto each spread plate </li><br />
</ol><br />
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<br />
<br />
<a name="RD"></a><br />
<br /> <br /> <br />
<br />
<h2 style="color:#0066CC">Restriction digest</h2><br />
<br />
<br /> <br />
<br />
<p>This protocol is part of the Construction Protocol. Start by selecting one of the parts you wish to combine as the vector <br />
(the plasmid will be kept) and the other part as the insert. The two parts will need to be mixed separately at the beginning.<br />
The parts must be kept separate for the digestion period.</p><br />
<br />
<br /><br />
<br />
<i>Add to the Insert Tube:</i><br />
<ul><br />
<li> 600 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> H<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
<br />
</ul><br />
<br />
<i> Add to the Vector Tube:</i><br />
<ul><br />
<li> 250 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of appropriate 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> HH<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
</ul><br />
<br />
<ol><br />
<li> Mix two tubes as indicated above </li><br />
<li> Put the tubes into a 37&deg;C water bath for one hour</li><br />
<li> Place the tubes into an 80&deg;C heating block for 20 minutes to heat-kill the enzymes in the tube</li><br />
<li> Freeze the parts until they are needed</li><br />
</ol><br />
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<br />
<a name="Ligation"></a><br />
<br /><br /> <h2 style="color:#0066CC">Ligation</h2><br />
<br /><br />
<p>This protocol is part of the Construction Protocol. The tubes from the Restriction Digest should be removed from the freezer and thawed on ice before beginning ligation.</p><br />
<br />
<ol><br />
<li> Mix 5 &micro;L of the insert and 5 &microl: of the vector in a new tube</li><br />
<li> Clearly label the tubes as unligated, write the date and freeze the tubes in -20&deg;C in case the transformation does not work </li><br />
<li> Add 10 &micro;L of 2x Quick Ligase Buffer and 1 &micro;L of Quick Ligase to the tube containing the mixed Insert and Vector </li><br />
<li> Let the tube sit at room temperature for 5 minutes</li><br />
<li> Transform this mix (all 21 &micro;L) into Top10 Competent Cells</li><br />
</ol><br />
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<br />
<a name="MPP"></a><br />
<br /><br /> <h2 style="color:#0066CC">Miniprep Plasmid Preparation (GenElute)</h2> <br />
<br /><br />
<p>This protocol is taken from the Sigma Aldrich distributed GenElute Miniprep Plasmid Preparation Kits. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer.<br />
We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.</p><br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4&deg;C</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet</li><br />
<li> Resuspend the pellet in 200 &micro;L of Resuspension Solution (with RNase A added) which is provided from the kit</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 200 &micro;L of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes</li><br />
<li> Add 350 &micro;L of Neutralization Solution and invert the tube 4-6 times to mix</li><br />
<li> Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate</li><br />
<li> Add 500 &micro;L of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube</li><br />
<li> Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid</li><br />
<li> Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube</li><br />
<li> Add 750 &micro;L of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column</li><br />
<li> Transfer the column to a new 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 50 &micro;L of double distilled water to the column and spin for 1 minute at 14 000 rpm</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
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<br />
<a name="AGE"></a><br />
<br /><br /><h2 style="color:#0066CC">Agarose Gel Electrophoresis</h2><br />
<br /><br />
<p>This procedure is involved in tandem with a PCR. The first step involves the making of an agarose gel as indicated by the Making of Agarose Gel protocol.</p><br />
<br />
<ol><br />
<li> Create an agarose gel tray</li><br />
<li> Place the gel tray into a gel dock where it will be run. Add TAE buffer such that the entire gel is covered</li><br />
<li> Make mixed tubes with 3 &micro;L DNA, 2 &micro;L Loading dye, and 15 &micro;L water</li><br />
<li> Insert 10 &micro;L of the mixture into each well with 5 &micro;L of the 1KB ladder in the first hole</li><br />
<li> Place thecovering on top and set it to run at 90V</li><br />
<li> When the bands are approximately halfway through the gel, which should be around 35-40 minutes, turn off the electricity and remove the gel</li><br />
<li> Use a computer imager to take a picture of the gel</li><br />
</ol><br />
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<br />
<a name="MOAG"></a><br />
<br /><br /> <h2 style="color:#0066CC">Making Agarose gel</h2><br />
<br /><br />
<p>The agarose gel is made in order to do agarose gel electrophoresis. The procedure is modified by the quantity of agarose added to the solution. The procedure here will detail the making of a 1.5% gel.</p><br />
<br />
<ol><br />
<li> Measure out 1.5g of agarose</li><br />
<li> Add the agarose to 100 mL of TAE buffer. COver the beaker with saran wrap with a hole punched in it</li><br />
<li> Microwave the solution for 30 seconds and then swirl. Then, microwave for 1 minute at high power and swirl. Finally, microwave for 1 minute further and swirl</li><br />
<li> Take this solution to the fume hood and add 3 &micro;L of ethidium bromide. Ethidium bromide is a suspected carcinogen so handle with care</li><br />
<li> Swirl the solution to allow the ethidium bromide to mix</li><br />
<li> Pour the solution into the tray. Use a 10 &micro;L pipette tip to pop any bubbles that may result and insert the comb</li><br />
<li> Allow the solution to solidify and remove the comb</li><br />
</ol><br />
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<br />
<a name="RORD"></a><br />
<br /><br /> <h2 style="color:#0066CC">Rehydration of registry DNA</h2><br />
<br /><br />
<p>The open source Parts Registry is where all parts are sent by iGEM teams each year. The distribution is done in three 384-well plates in dry DNA form.<br />
The DNA must be rehydrated, transformed into Top10 Competent cells and then plasmid prepped using the Miniprep Plasmid Prep Protocol as listed above before they are in usable DNA form.</p><br />
<br />
<ol><br />
<li> Use a 10 &micro;L pipette tip to puncture the aluminium foil covering of the desired well of DNA</li><br />
<li> After 5 minutes to allow the DNA to thaw, add 10 &micro;L of double distilled water to the well and pipette up and down 3-4 times until the liquid comes up red</li><br />
<li> Take 2 &micro;L of DNA and transform these into Top10 Competent Cells using the Transformation Protocol</li><br />
</ol><br />
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<br />
<a name="CT"></a><br />
<br /><br /> <h2 style="color:#0066CC">Construction technique</h2><br />
<br /><br />
<p>This protocol combines the Restriction Digest with the Ligation protocol as well as adding Antarctic Phosphatase Protocol.</p><br />
<br />
<br /> <u>Restriction Digest</u><br />
<br />
<br /><br />
<p><i>Add to the Insert Tube:</i></p><br />
<ul><br />
<li> 600 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> H<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
<br />
</ul><br />
<br />
<p><i> Add to the Vector Tube:</i></p><br />
<ul><br />
<li> 250 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of appropriate 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> HH<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
</ul><br />
<br />
<ol><br />
<li> Mix two tubes as indicated above </li><br />
<li> Put the tubes into a 37&deg;C water bath for one hour</li><br />
<li> Place the tubes into an 80&deg;C heating block for 20 minutes to heat-kill the enzymes in the tube</li><br />
<li> Freeze the parts until they are needed</li><br />
</ol><br />
<br />
<br/><p><u> Ligation Protocol with Antarctic Phosphatase </u></p><br />
<br />
<ol><br />
<li> Mix 5 &micro;L of the insert and 5 &microl: of the vector in a new tube</li><br />
<li> Clearly label the tubes as unligated, write the date and freeze the tubes in -20&deg;C in case the transformation does not work </li><br />
<li> Add 10 &micro;L of 2x Quick Ligase Buffer and 1 &micro;L of Quick Ligase to the tube containing the mixed Insert and Vector </li><br />
<li> Let the tube sit at room temperature for 5 minutes</li><br />
<li> Add 5 &micro;L of 10x Antarctic Phosphatase Buffer, 4 &micro;L of water, and 1 &micro;L of Antarctic Phosphatase to the Vector while freezing the insert. Put the tube into a 37&deg;C water bath for 30 minutes and then place into the 65 &deg;C heating block for 10 minutes</li><br />
<li> Transform this mix (all 21 &micro;L) into Top10 Competent Cells</li><br />
</ol><br />
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<br />
<a name="OC"></a><br />
<br /><br /> <h2 style="color:#0066CC">Overnight cultures</h2><br />
<br /><br />
<p>This procedure is used before the Sigma Aldrich GenElute Plasmid Preparation. You will need a 10 mL culture tube, 5 mL of LB Broth, Antibiotic, and single colonies on a plate.</p><br />
<br />
<ol><br />
<li> Pipette 5 mL of LB Broth into the culture tube. </li><br />
<li> Add Antibiotic (10 &micro;L Ampicillin, 5 &micro;L Kanamycin, or 3 &micro;L Chloramphenicol)</li><br />
<li> Select a single colony using a 200 &micro;L sterile pipette tip</li><br />
<li> Place the culture tube into the shaker and let it shake at 175 rpm at 37&deg;C</li><br />
</ol><br />
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<br />
<a name="GSP"></a><br />
<br /><br /><h2 style="color:#0066CC">Glycerol stock preparation</h2><br />
<br /><br />
<p>This procedure is used to make long term stocks of plasmid parts that will definitely be used later on. The procedure was given by our lab technician Deirdre Lobb.</p><br />
<br />
<ol><br />
<li> Grow 5 mL overnight cultures for the bacteria containing the plasmid which you wish to create glycerol stocks of (This procedure is indicated by the Overnight Growth Protocol)</li><br />
<li> Take 1 mL of the culture and add it to 1 mL of autoclaved 50% glycerol</li><br />
<li> Divide this solution into two tubes holding 1 mL each and store these in 1.5 mL microcentrifuge tubes</li><br />
<li> Use dry ice to flash freeze the tubes and store the glycerol stocks in a -80&deg;C freezer</li><br />
<br />
</ol><br />
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<br />
<a name="LAPPP"></a><br />
<br /><br /> <h2 style="color:#0066CC">LB agar preparation</h2><br />
<br />
<ol><br />
<li> Autoclave mixture of dH2O and agar</li><br />
<li> Add either 1 mL of Ampicillin, 0.5 mL Kanamycin or 0.35 mL Chloramphenicol</li><br />
<li> Pour plates, flame and mark plates</li><br />
<li> Let dry overnight</li><br />
<br />
</ol><br />
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<br />
<a name="QSMK"></a><br />
<br /><br /> <h2 style="color:#0066CC">QIAprep spin Miniprep kit </h2><br />
<br /><br />
<p>This protocol is taken from the Qiagen distributed QIAprep Spin Miniprep Kit. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer.<br />
We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.</p><br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4&deg;C</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet</li><br />
<li> Resuspend the pellet in 250 &micro;L of Buffer P1 (with RNase A added) which is provided from the kit</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 250 &micro;L of Buffer P2 and invert gently to mix. Allow the mixture to clear for less than 5 minutes</li><br />
<li> Add 350 &micro;L of Buffer N3 and invert the tube 4-6 times to mix</li><br />
<li> Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate</li><br />
<li> Add 500 &micro;L of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube</li><br />
<li> Transfer the lysate into the QIAprep spin column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid</li><br />
<li> Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube</li><br />
<li> Add 750 &micro;L of Buffer PE with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column</li><br />
<li> Transfer the column to a new 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 50 &micro;L of double distilled water to the column and spin for 1 minute at 14 000 rpm</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
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<br />
<br />
<a name="GE"></a><br />
<br /><br /> <h2 style="color:#0066CC">Gel extraction</h2><br />
<br /><br />
<p>This protocol is utilized in accordance to the manufacturer's protocol from Omega E.Z.N.A (EaZy Nucleic Acid Isolation)</p><br />
<br />
<ol><br />
<li> Place gel on the UV box</li><br />
<li> Carefully extract the fragment suspended in the gel>/li><br />
<li> Mass gel fragments </li><br />
<li> Place fragment into a 1.5 mL tube and add 4 &micro;L of H2O </li><br />
<li> Volume of water added to volume of gel is 200% however if fragment it small 1 mL of water will suffice </li><br />
<li> Remove H2O </li><br />
<li> Add equal amounts of H2O and Binding Buffer (XP2) to the gel</li><br />
<li> Incubate mixture at 55 degrees for 7 mins </li><br />
<li> Mix with vortex for 2 mins </li><br />
<li> Place in the HiBind DNA Mini Column in the 2 mL tube </li><br />
<li> Add 700 &micro;L at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Add 300 &micro;L Binding Buffer (XP2) into the HiBind DNA Mini Column and spin down at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Wash the column with 700 &micro;L of SPW buffer with added ethanol and spin down at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Wash the column with 700 &micro;L of SPW buffer again and spin down at 10,000xg for 1 min </li><br />
<li> Discard the liquid </li><br />
<li> Spin down the column at 13,000xg for 1 min to dry the column </li><br />
<li> Elute in 50 &micro;L of H2O and wait 1 min </li><br />
<li> Spin down the column at 13,000xg for 1 min to dry the column </li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
<p><a href="JavaScript:newPopup('https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols/Comments#GE');">The Theory</a> - <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#top">Back to top</a></p><br />
<br />
<br />
<br />
<a name="PP"></a><br />
<br /><br /><h2 style="color:#0066CC">PCR purification (Vaccum)</h2><br />
<br /><br />
<p>This protocol is utilized in accordance to the manufacturer's protocol from Qiagen PCR Vacuum Prep Kit </p><br />
<ol><br />
<li> Add PCR product</li><br />
<li> Distribute liquid evenly by evenly tapping</li><br />
<li> Vacuum for 15-20 mins to dry</li><br />
<li> Add 20 &micro;L H2O</li><br />
<li> Tap lightly against the table to redistribute the H2O on the surface of the well</li><br />
<li> Pipette contents and transfer to clean 1.5 mL tube</li><br />
</ol><br />
<p><a href="JavaScript:newPopup('https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols/Comments#PP');">The Theory</a> - <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#top">Back to top</a></p><br />
</div><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/Future_DirectionsTeam:Calgary/Notebook/Future Directions2010-10-27T10:30:10Z<p>Pauladamiak: </p>
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<span id="bodytitle"><h1>Future Directions</h1></span><br />
<br />
<h3>Wetlab</h3><br />
<br />
<p>The protein expression detection kit is designed such that one can clone in their gene of interest (GOI) into the kit using a simple biobrick cloning method and the system will give a simple visual output that would indicate the point of protein expression that is failing. For example: if the output is red that indicates that the protein is misfolding in the periplasm, if there is green flouroscent protein expressed, it indicates that protein is misfolding in the cytoplasm.</p><br />
<p>This kit employs existing reporter coupling methods to make a compact kit which detects problems in one step. To take this kit a step further, the team will be working on adding more promoters in the system so that misfolding stress can be detected at different levels both high and low. This would allow the system to be more sensitive to different levels of protein folding stress. We also plan on including different coloured reporters to indicate which system got activated. For example: currently we are using GFP for the sigma32 system which detects cytoplasmic stress and RFP for the Cpx regulon which detects periplasmic stress. In the future, we can couple the Lol system which monitors outer membrane lipoprotein with YFP, the Ppi system which targets outer membrane Beta-barrels with CFP and so on. This allows the system to be more diverse and specific, because systems like Lol and Ppi have very specific target substrates. </p><br />
<p>Another interesting future direction with this project is building an auto-tuner with the stress detector. Literature has established that proteins at low levels fold properly, however at high levels, which are used during synthetic protein production, they misfold due to high protein concentration. The autotuner would be included in the current system to help control the level of protein expression such that if the protein misfolds, the stress promoter turns on which in turns represses the expression of the GOI until it does not misfolds or form inclusion bodies. This would allow artificial expression of the GOI successfully such that the protein of interest (POI) can be collected.</p><br />
<br />
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-7.png" border="0" alt="auto tuner"></a><br />
<br />
<p>Schematic of the auto-tuner<br />
1. Constitutive production of the GOI using a TetR promoter.<br />
2. Activation of Stress promoter due to overproduction of GOI and misfolding of GOI.<br />
3. Production of Tet product and the repression of the TetR promoter as a result.<br />
</p><br />
<br />
<br />
<h3>Modeling</h3><br />
<br />
<p>The modeling project currently consists of examining different parameters such as temperature, pH and hydrophobic content of a protein in order to generate a ratio of inclusion body compared to folded proteins. Future directions for the modeling project would be incorporate more variables. One of the interesting variables to include would be protein motifs such as protease domains. This approach would make the model more diverse because it would account for the fact that every protein is different. However, this would also be a generalization about certain stretches of amino acids and be further criticized by proteomics expert as it is too vague and might not always hold. Another interesting future direction would be to create software or a user interphase that is much easier to operate than MATLAB, where the parameters could be changed easily and the input that would be required would be pI (isoelectric point) of the protein and the amino acid sequence of the protein. This could contribute to a software entry for iGEM next year if the project is continued and successfully finished. </p><br />
<p><br />
Currently, the model uses previously established models in order to contribute to the algorithm that generates the ratio. In the future, we would like to incorporate the use of Markov models which would account for the random chance of events happening and probabilities a lot better than the current algorithms that are being used. </p><br />
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<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
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<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
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<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety"><img src="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"></a></img></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
</tr><br />
</table><br />
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<br />
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<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
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<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety"><img src="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"></a></img></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
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<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
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<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"><img src="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"></a></img></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/NotebookTeam:Calgary/Notebook2010-10-27T10:25:21Z<p>Pauladamiak: </p>
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<br />
<div class="container"><br />
<br />
<div class="sidebar"><br />
<br />
<h1>Sections</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"><img src="https://static.igem.org/mediawiki/2010/3/37/Safety1.png" /></a></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/NotebookTeam:Calgary/Notebook2010-10-27T10:23:36Z<p>Pauladamiak: </p>
<hr />
<div>{{CalgaryMenu}}<br />
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</head><br />
<br />
<body><br />
<br />
<div class="container"><br />
<br />
<div class="sidebar"><br />
<br />
<h1>Sections</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"><img src="https://static.igem.org/mediawiki/2010/3/37/Safety_button.png" /></a></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/File:Safety1.pngFile:Safety1.png2010-10-27T10:22:35Z<p>Pauladamiak: uploaded a new version of "Image:Safety1.png"</p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/NotebookTeam:Calgary/Notebook2010-10-27T10:18:54Z<p>Pauladamiak: </p>
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<div>{{CalgaryMenu}}<br />
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<style><br />
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div.container{<br />
background-image:url("http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/PageContainer-1.png");<br />
background-repeat: repeat-y;<br />
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} <br />
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div.sidebar{<br />
width: 235px;<br />
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<h1>Sections</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://static.igem.org/mediawiki/2010/3/39/Safety1.png"><img src="https://static.igem.org/mediawiki/2010/3/37/Safety_button.png"></a></img></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/File:Safety1.pngFile:Safety1.png2010-10-27T10:18:24Z<p>Pauladamiak: uploaded a new version of "Image:Safety1.png"</p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/File:Safety1.pngFile:Safety1.png2010-10-27T10:16:45Z<p>Pauladamiak: </p>
<hr />
<div></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/NotebookTeam:Calgary/Notebook2010-10-27T10:15:00Z<p>Pauladamiak: </p>
<hr />
<div>{{CalgaryMenu}}<br />
<br />
<html><br />
<head><br />
<br />
<style><br />
<br />
div.container{<br />
background-image:url("http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/PageContainer-1.png");<br />
background-repeat: repeat-y;<br />
width: 975px;<br />
float: left;<br />
margin-left: -5px;<br />
margin-bottom: 20px;<br />
} <br />
<br />
div.sidebar{<br />
width: 235px;<br />
float: left;<br />
margin-left: 14px;<br />
margin-right: 10px;<br />
padding-left: 0px;<br />
margin-top: 10px;<br />
<br />
}<br />
<br />
div.sidebar h1, div.sidebar ul li a{<br />
color:#7b54cc;<br />
}<br />
<br />
<br />
div.mainbody{<br />
width: 670px;<br />
float: left;<br />
margin-left: 20px;<br />
margin-right: 20px;<br />
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}<br />
<br />
#bodytitle h1{<br />
color:#ff8400;<br />
}<br />
<br />
.blue p, .blue a{<br />
color:#54bcf8;<br />
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.green p, .green a{<br />
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}<br />
<br />
div.mainbody table tr td img{<br />
padding-right: 15px;<br />
padding-bottom: 10px;<br />
}<br />
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</style><br />
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</head><br />
<br />
<body><br />
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<div class="container"><br />
<br />
<div class="sidebar"><br />
<br />
<h1>Sections</h1><br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
</ul><br />
<br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
</tr><br />
<br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
</td><br />
</tr><br />
<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety"><img src="https://static.igem.org/mediawiki/2010/3/37/Safety_button.png"></a></img></td><br />
<td><span style="color:#FFCC33"><p>Safety</p></span><br />
<p>To see our completed iGEM safety requirements questionnaire follow this link: <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety">safety questionnaire</a> <br />
</p><br />
</td><br />
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</html></div>Pauladamiakhttp://2010.igem.org/File:Safety_button.pngFile:Safety button.png2010-10-27T10:10:18Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
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<span id="bodytitle"><h1>Team Notebook</h1></span><br />
<br />
<p>The University of Calgary 2010 iGEM team keeps a record of our summer's work in the Notebook. The Notebook contains daily activities, a log of our brainstorming sessions and meetings, and a handy, detailed reference guide to each lab procedure we have used.</p><br />
<br />
<table><br />
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<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/CalendarIcon.png"></a></img></td><br />
<td><span class="blue"><p>Calendar</p></span><br />
<p>Want to know what we've been up to? Click <span class="blue"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">here</a></span> for a record of each team member's activity over the summer.</p><br />
</td><br />
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<br />
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<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/BrainstormIcon.png"></a></img></td><br />
<td><span class="green"><p>Future Directions</p></span><br />
<p>With this project, there are tons of directions that we can head. Click <span class="green"><a href="https://2010.igem.org/Team:Calgary/Notebook/Brainstorming">here</a></span> to see some of the ideas we have in store for the future.</p><br />
</td><br />
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<tr><br />
<td><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ProtocolIcon.png"></a></img></td><br />
<td><span class="purple"><p>Safety and Protocols</p></span><br />
<p>So you've looked at our <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">calendar</a></span>. That's all well and good... but what is this "PCR" thing that keeps popping up? If you want to see details for each lab procedure mentioned in our calendar, click <span class="purple"><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">here</a></span> for a reference.</p><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/Future_DirectionsTeam:Calgary/Notebook/Future Directions2010-10-27T09:38:13Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a></li><br />
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<br />
<span id="bodytitle"><h1>Future Directions</h1></span><br />
<br />
<h3>Wetlab</h3><br />
<br />
<p>The protein expression detection kit is designed such that one can clone in their gene of interest (GOI) into the kit using a simple biobrick cloning method and the system will give a simple visual output that would indicate the point of protein expression that is failing. For example: if the output is red that indicates that the protein is misfolding in the periplasm, if there is green flouroscent protein expressed, it indicates that protein is misfolding in the cytoplasm.</p><br />
<p>This kit employs existing reporter coupling methods to make a compact kit which detects problems in one step. To take this kit a step further, the team will be working on adding more promoters in the system so that misfolding stress can be detected at different levels both high and low. This would allow the system to be more sensitive to different levels of protein folding stress. We also plan on including different coloured reporters to indicate which system got activated. For example: currently we are using GFP for the sigma32 system which detects cytoplasmic stress and RFP for the Cpx regulon which detects periplasmic stress. In the future, we can couple the Lol system which monitors outer membrane lipoprotein with YFP, the Ppi system which targets outer membrane Beta-barrels with CFP and so on. This allows the system to be more diverse and specific, because systems like Lol and Ppi have very specific target substrates. </p><br />
<p>Another interesting future direction with this project is building an auto-tuner with the stress detector. Literature has established that proteins at low levels fold properly, however at high levels, which are used during synthetic protein production, they misfold due to high protein concentration. The autotuner would be included in the current system to help control the level of protein expression such that if the protein misfolds, the stress promoter turns on which in turns represses the expression of the GOI until it does not misfolds or form inclusion bodies. This would allow artificial expression of the GOI successfully such that the protein of interest (POI) can be collected.</p><br />
<br />
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=Untitled-7.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/Untitled-7.png" border="0" alt="auto tuner"></a><br />
<br />
<p>Schematic of the auto-tuner<br />
1. Constitutive production of the GOI using a TetR promoter.<br />
2. Activation of Stress promoter due to overproduction of GOI and misfolding of GOI.<br />
3. Production of Tet product and the repression of the TetR promoter as a result.<br />
</p><br />
<br />
<br />
<h3>Modeling</h3><br />
<br />
<p>The modeling project currently consists of examining different parameters such as temperature, pH and hydrophobic content of a protein in order to generate a ratio of inclusion body compared to folded proteins. Future directions for the modeling project would be incorporate more variables. One of the interesting variables to include would be protein motifs such as protease domains. This approach would make the model more diverse because it would account for the fact that every protein is different. However, this would also be a generalization about certain stretches of amino acids and be further criticized by proteomics expert as it is too vague and might not always hold. Another interesting future direction would be to create software or a user interphase that is much easier to operate than MATLAB, where the parameters could be changed easily and the input that would be required would be pI (isoelectric point) of the protein and the amino acid sequence of the protein. This could contribute to a software entry for iGEM next year if the project is continued and successfully finished. </p><br />
<p><br />
Currently, the model uses previously established models in order to contribute to the algorithm that generates the ratio. In the future, we would like to incorporate the use of Markov models which would account for the random chance of events happening and probabilities a lot better than the current algorithms that are being used. </p><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Notebook/Safety_And_ProtocolsTeam:Calgary/Notebook/Safety And Protocols2010-10-27T09:36:52Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Calendar">Calendar</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Future_Directions">Future Directions</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols">Protocols</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#PCR">Taq Polymerase Chain Reaction</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#TCCIP">Transformable Competent Cell Induction Protocol</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#BTP">Bacterial Transformation Protocol </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#RD">Restriction Digest</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#Ligation">Ligation</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#MPP">Miniprep Plasmid Preparation (GenElute</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#AGE">Agarose Gel Electrophoresis </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#MOAG">Making of Agarose Gel </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#RORD">Rehydration of Registry DNA</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#CT">Construction Technique</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#OC">Overnight Cultures</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#GSP">Glycerol Stock Preparation </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#LAPPP">LB Agar Plate Preparation Protocol </a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#QSMK">QIAprep Spin Miniprep Kit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#GE">Gel Extraction</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#PP">PCR Purification (Vacuum)</a></li><br />
</ul><br />
</li><br />
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<a name="top"></a><br />
<span id="bodytitle"><h1>iGEM Laboratory Procedures</h1></span><br />
<br />
<a name="PCR"></a><br />
<h2 style="color:#0066CC">Taq polymerase chain reaction</h2><br />
<br />
<i> <br /> <br /> Polymerase Chain Reaction Master Mix </i><br />
<br />
<br />
<table border="2"><br />
<tr><br />
<td> <b> Reagent </b> </td><br />
<td> <b> 1x Mix (&micro;L) </b> </td><br />
<td> <b> 5x Mix (&micro;L) </b> </td><br />
</tr><br />
<br />
<tr><br />
<td> H<sub>2</sub>O </td><br />
<td> 28 </td><br />
<td> 140 </td><br />
</tr><br />
<br />
<tr><br />
<td> 10x Buffer </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> 2 mM dNTP </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> Forward Primer (2 mM) </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> Reverse Primer (2 mM) </td><br />
<td> 5 </td><br />
<td> 25 </td><br />
</tr><br />
<br />
<tr><br />
<td> 50 mM MgCl<sub>2</sub> </td><br />
<td> 1.5 </td><br />
<td> 7.5 </td><br />
</tr><br />
<br />
<tr><br />
<td> Taq Polymerase </td><br />
<td> 0.5 </td><br />
<td> 2.5 </td><br />
</tr><br />
<br />
<tr><br />
<td> <b> Total </b> </td><br />
<td> <b> 50 </b> </td><br />
<td> <b> 250 </b> </td> <br />
</tr><br />
</table><br />
<br />
<br /><br />
<br />
<p>DNA template is required. The quantity of water decreases in proportion to the volume of DNA template added so that the total volume remains the same.<p><br />
<br />
<br /> <i> Thermocycler PCR Program </i><br />
<br />
<table border="2"><br />
<tr><br />
<td> <b> Temperature (&deg;C)</b> </td><br />
<td> <b> Time (mins) </b> </td><br />
</tr><br />
<br />
<tr><br />
<td> 95.0 </td><br />
<td> 5:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 95.0 </td><br />
<td> 1:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 55.0* </td><br />
<td> 0:30 </td><br />
</tr><br />
<br />
<tr><br />
<td> 72.0 </td><br />
<td> 1:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 72.0 </td><br />
<td> 10:00 </td><br />
</tr><br />
<br />
<tr><br />
<td> 4.0 </td><br />
<td> &infin; </td><br />
</tr><br />
<br />
</table> <br />
<br />
<br /><br />
<p> <b> NOTE: </b> * indicates that the temperature of the step is primer specific.<br />
Steps 2 through 4 are repeated 30 times.<br />
The length of Step 4 is 1 minute for every 1000 base pairs of the template to be amplified.</p><br />
<p><a href="JavaScript:newPopup('https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols/Comments#PCR');">The Theory</a> - <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#top">Back to top</a></p><br />
<br />
<br />
<a name="TCCIP"></a><br />
<br /> <br /> <h2 style="color:#0066CC">Making competent cells</h2><br />
<br />
<br /> <p>This procedure was done using Top10 Competent cells ordered from Invitrogen. 50 mL Falcon tubes were used for this protocol.</p><br />
<br />
<ol><br />
<li> Innoculate 5-10 mL LB at 37&deg;C while shaking </li><br />
<li> Subculture 1 mL of bacteria solution into 50 mL LB broth at 37&deg; while shaking<br />
until OD600 is 0.4-0.6 (This step should require approximately 2.5 hours) </li><br />
<li> Centrifuge the subculture at 10 000 rpm at 4&deg;C for 2 minutes </li><br />
<li> Resuspend pellet in 12.5 mL of cold CaCl<sub>2</sub> (50 mM) and leave on ice for 10 minutes </li><br />
<li> Centrifuge at 10 000 rpm at 4&deg;C for 2 minutes and resuspend in 2 mL of cold CaCl<sub>2</sub> (50 mM, 15% glycerol solution) </li><br />
<li> Leave on ice for at least 30 minutes and then aliquot 200 uL and freeze at -80&deg;C </li><br />
</ol><br />
<p><a href="JavaScript:newPopup('https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols/Comments#TCCIP');">The Theory</a> - <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#top">Back to top</a></p><br />
<br />
<a name="BTP"></a><br />
<br /> <br /> <br />
<br />
<h2 style="color:#0066CC">Bacterial transformation</h2><br />
<br />
<br /> <br />
<ol> <br />
<li> Thaw Competent Cells </li> <br />
<li> Add 10-100 ng of DNA </li><br />
<li> Ice solution for 30 minutes </li><br />
<li> Heat shock solution (5 minutes at 37&deg;C or 2 minutes at 42&deg;C) </li><br />
<li> Ice solution for 5 minutes </li><br />
<li> Recover with 250 &micro;L of SOC (30 minutes for Ampicilin resistant plasmids and 60 minutes for kanamycin resistant plasmids) </li><br />
<li> Centrifuge for 5 seconds at 14 000 rpm and concentrate solution to 100 μL </li><br />
<li> Plate 20-50 &micro;L onto each spread plate </li><br />
</ol><br />
<p><a href="JavaScript:newPopup('https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols/Comments#BTP');">The Theory</a> - <a href="https://2010.igem.org/Team:Calgary/Notebook/Safety_And_Protocols#top">Back to top</a></p><br />
<br />
<br />
<a name="RD"></a><br />
<br /> <br /> <br />
<br />
<h2 style="color:#0066CC">Restriction digest</h2><br />
<br />
<br /> <br />
<br />
<p>This protocol is part of the Construction Protocol. Start by selecting one of the parts you wish to combine as the vector <br />
(the plasmid will be kept) and the other part as the insert. The two parts will need to be mixed separately at the beginning.<br />
The parts must be kept separate for the digestion period.</p><br />
<br />
<br /><br />
<br />
<i>Add to the Insert Tube:</i><br />
<ul><br />
<li> 600 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> H<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
<br />
</ul><br />
<br />
<i> Add to the Vector Tube:</i><br />
<ul><br />
<li> 250 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of appropriate 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> HH<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
</ul><br />
<br />
<ol><br />
<li> Mix two tubes as indicated above </li><br />
<li> Put the tubes into a 37&deg;C water bath for one hour</li><br />
<li> Place the tubes into an 80&deg;C heating block for 20 minutes to heat-kill the enzymes in the tube</li><br />
<li> Freeze the parts until they are needed</li><br />
</ol><br />
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<br />
<a name="Ligation"></a><br />
<br /><br /> <h2 style="color:#0066CC">Ligation</h2><br />
<br /><br />
<p>This protocol is part of the Construction Protocol. The tubes from the Restriction Digest should be removed from the freezer and thawed on ice before beginning ligation.</p><br />
<br />
<ol><br />
<li> Mix 5 &micro;L of the insert and 5 &microl: of the vector in a new tube</li><br />
<li> Clearly label the tubes as unligated, write the date and freeze the tubes in -20&deg;C in case the transformation does not work </li><br />
<li> Add 10 &micro;L of 2x Quick Ligase Buffer and 1 &micro;L of Quick Ligase to the tube containing the mixed Insert and Vector </li><br />
<li> Let the tube sit at room temperature for 5 minutes</li><br />
<li> Transform this mix (all 21 &micro;L) into Top10 Competent Cells</li><br />
</ol><br />
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<br />
<a name="MPP"></a><br />
<br /><br /> <h2 style="color:#0066CC">Miniprep Plasmid Preparation (GenElute)</h2> <br />
<br /><br />
<p>This protocol is taken from the Sigma Aldrich distributed GenElute Miniprep Plasmid Preparation Kits. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer.<br />
We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.</p><br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4&deg;C</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet</li><br />
<li> Resuspend the pellet in 200 &micro;L of Resuspension Solution (with RNase A added) which is provided from the kit</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 200 &micro;L of Lysis Solution and invert gently to mix. Allow the mixture to clear for less than 5 minutes</li><br />
<li> Add 350 &micro;L of Neutralization Solution and invert the tube 4-6 times to mix</li><br />
<li> Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate</li><br />
<li> Add 500 &micro;L of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube</li><br />
<li> Transfer the lysate into the binding column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid</li><br />
<li> Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube</li><br />
<li> Add 750 &micro;L of Wash Solution with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column</li><br />
<li> Transfer the column to a new 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 50 &micro;L of double distilled water to the column and spin for 1 minute at 14 000 rpm</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
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<br />
<a name="AGE"></a><br />
<br /><br /><h2 style="color:#0066CC">Agarose Gel Electrophoresis</h2><br />
<br /><br />
<p>This procedure is involved in tandem with a PCR. The first step involves the making of an agarose gel as indicated by the Making of Agarose Gel protocol.</p><br />
<br />
<ol><br />
<li> Create an agarose gel tray</li><br />
<li> Place the gel tray into a gel dock where it will be run. Add TAE buffer such that the entire gel is covered</li><br />
<li> Make mixed tubes with 3 &micro;L DNA, 2 &micro;L Loading dye, and 15 &micro;L water</li><br />
<li> Insert 10 &micro;L of the mixture into each well with 5 &micro;L of the 1KB ladder in the first hole</li><br />
<li> Place thecovering on top and set it to run at 90V</li><br />
<li> When the bands are approximately halfway through the gel, which should be around 35-40 minutes, turn off the electricity and remove the gel</li><br />
<li> Use a computer imager to take a picture of the gel</li><br />
</ol><br />
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<br />
<a name="MOAG"></a><br />
<br /><br /> <h2 style="color:#0066CC">Making Agarose gel</h2><br />
<br /><br />
<p>The agarose gel is made in order to do agarose gel electrophoresis. The procedure is modified by the quantity of agarose added to the solution. The procedure here will detail the making of a 1.5% gel.</p><br />
<br />
<ol><br />
<li> Measure out 1.5g of agarose</li><br />
<li> Add the agarose to 100 mL of TAE buffer. COver the beaker with saran wrap with a hole punched in it</li><br />
<li> Microwave the solution for 30 seconds and then swirl. Then, microwave for 1 minute at high power and swirl. Finally, microwave for 1 minute further and swirl</li><br />
<li> Take this solution to the fume hood and add 3 &micro;L of ethidium bromide. Ethidium bromide is a suspected carcinogen so handle with care</li><br />
<li> Swirl the solution to allow the ethidium bromide to mix</li><br />
<li> Pour the solution into the tray. Use a 10 &micro;L pipette tip to pop any bubbles that may result and insert the comb</li><br />
<li> Allow the solution to solidify and remove the comb</li><br />
</ol><br />
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<br />
<a name="RORD"></a><br />
<br /><br /> <h2 style="color:#0066CC">Rehydration of registry DNA</h2><br />
<br /><br />
<p>The open source Parts Registry is where all parts are sent by iGEM teams each year. The distribution is done in three 384-well plates in dry DNA form.<br />
The DNA must be rehydrated, transformed into Top10 Competent cells and then plasmid prepped using the Miniprep Plasmid Prep Protocol as listed above before they are in usable DNA form.</p><br />
<br />
<ol><br />
<li> Use a 10 &micro;L pipette tip to puncture the aluminium foil covering of the desired well of DNA</li><br />
<li> After 5 minutes to allow the DNA to thaw, add 10 &micro;L of double distilled water to the well and pipette up and down 3-4 times until the liquid comes up red</li><br />
<li> Take 2 &micro;L of DNA and transform these into Top10 Competent Cells using the Transformation Protocol</li><br />
</ol><br />
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<br />
<a name="CT"></a><br />
<br /><br /> <h2 style="color:#0066CC">Construction technique</h2><br />
<br /><br />
<p>This protocol combines the Restriction Digest with the Ligation protocol as well as adding Antarctic Phosphatase Protocol.</p><br />
<br />
<br /> <u>Restriction Digest</u><br />
<br />
<br /><br />
<p><i>Add to the Insert Tube:</i></p><br />
<ul><br />
<li> 600 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> H<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
<br />
</ul><br />
<br />
<p><i> Add to the Vector Tube:</i></p><br />
<ul><br />
<li> 250 ng of DNA (Calculate this from the concentration of plasmid)</li><br />
<li> 3.5 &micro;L of appropriate 10x Buffer</li><br />
<li> 0.5 &micro;L of each restriction enzyme used (2 of EcoRI, XbaI, SpeI, or PstI)</li><br />
<li> HH<sub>2</sub>O such that the volume of water and DNA in the tube is 30.5 &micro;L and the total volume in each tube is 35 &micro;L</li><br />
</ul><br />
<br />
<ol><br />
<li> Mix two tubes as indicated above </li><br />
<li> Put the tubes into a 37&deg;C water bath for one hour</li><br />
<li> Place the tubes into an 80&deg;C heating block for 20 minutes to heat-kill the enzymes in the tube</li><br />
<li> Freeze the parts until they are needed</li><br />
</ol><br />
<br />
<br/><p><u> Ligation Protocol with Antarctic Phosphatase </u></p><br />
<br />
<ol><br />
<li> Mix 5 &micro;L of the insert and 5 &microl: of the vector in a new tube</li><br />
<li> Clearly label the tubes as unligated, write the date and freeze the tubes in -20&deg;C in case the transformation does not work </li><br />
<li> Add 10 &micro;L of 2x Quick Ligase Buffer and 1 &micro;L of Quick Ligase to the tube containing the mixed Insert and Vector </li><br />
<li> Let the tube sit at room temperature for 5 minutes</li><br />
<li> Add 5 &micro;L of 10x Antarctic Phosphatase Buffer, 4 &micro;L of water, and 1 &micro;L of Antarctic Phosphatase to the Vector while freezing the insert. Put the tube into a 37&deg;C water bath for 30 minutes and then place into the 65 &deg;C heating block for 10 minutes</li><br />
<li> Transform this mix (all 21 &micro;L) into Top10 Competent Cells</li><br />
</ol><br />
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<br />
<a name="OC"></a><br />
<br /><br /> <h2 style="color:#0066CC">Overnight cultures</h2><br />
<br /><br />
<p>This procedure is used before the Sigma Aldrich GenElute Plasmid Preparation. You will need a 10 mL culture tube, 5 mL of LB Broth, Antibiotic, and single colonies on a plate.</p><br />
<br />
<ol><br />
<li> Pipette 5 mL of LB Broth into the culture tube. </li><br />
<li> Add Antibiotic (10 &micro;L Ampicillin, 5 &micro;L Kanamycin, or 3 &micro;L Chloramphenicol)</li><br />
<li> Select a single colony using a 200 &micro;L sterile pipette tip</li><br />
<li> Place the culture tube into the shaker and let it shake at 175 rpm at 37&deg;C</li><br />
</ol><br />
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<br />
<a name="GSP"></a><br />
<br /><br /><h2 style="color:#0066CC">Glycerol stock preparation</h2><br />
<br /><br />
<p>This procedure is used to make long term stocks of plasmid parts that will definitely be used later on. The procedure was given by our lab technician Deirdre Lobb.</p><br />
<br />
<ol><br />
<li> Grow 5 mL overnight cultures for the bacteria containing the plasmid which you wish to create glycerol stocks of (This procedure is indicated by the Overnight Growth Protocol)</li><br />
<li> Take 1 mL of the culture and add it to 1 mL of autoclaved 50% glycerol</li><br />
<li> Divide this solution into two tubes holding 1 mL each and store these in 1.5 mL microcentrifuge tubes</li><br />
<li> Use dry ice to flash freeze the tubes and store the glycerol stocks in a -80&deg;C freezer</li><br />
<br />
</ol><br />
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<br />
<a name="LAPPP"></a><br />
<br /><br /> <h2 style="color:#0066CC">LB agar preparation</h2><br />
<br />
<ol><br />
<li> Autoclave mixture of dH2O and agar</li><br />
<li> Add either 1 mL of Ampicillin, 0.5 mL Kanamycin or 0.35 mL Chloramphenicol</li><br />
<li> Pour plates, flame and mark plates</li><br />
<li> Let dry overnight</li><br />
<br />
</ol><br />
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<br />
<a name="QSMK"></a><br />
<br /><br /> <h2 style="color:#0066CC">QIAprep spin Miniprep kit </h2><br />
<br /><br />
<p>This protocol is taken from the Qiagen distributed QIAprep Spin Miniprep Kit. We modified the elution portion of the procedure by using double distilled water to elute rather than using TE buffer.<br />
We also skipped the step with the optional wash solution. Instead, the step with the addition of Wash Solution in the Column Tube was done twice. Another company's kit was used occasionally to prep the plasmids but the two kits were never mixed.</p><br />
<br />
<ol><br />
<li> Make overnight cultures from LB agar plate growth (The protocol for the making of overnight cultures can be found as a separate protocol)</li><br />
<li> After allowing approximately 16 hours of growth, pellet the cells using a centrifuge for 20 minutes at a speed of 4000 rpm at 4&deg;C</li><br />
<li> Discard the supernatant, while being careful not to discard any of the pellet</li><br />
<li> Resuspend the pellet in 250 &micro;L of Buffer P1 (with RNase A added) which is provided from the kit</li><br />
<li> Transfer the solution from a Falcon tube to a 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 250 &micro;L of Buffer P2 and invert gently to mix. Allow the mixture to clear for less than 5 minutes</li><br />
<li> Add 350 &micro;L of Buffer N3 and invert the tube 4-6 times to mix</li><br />
<li> Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The resulting solution will be known as the lysate</li><br />
<li> Add 500 &micro;L of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the liquid underneath the binding tube</li><br />
<li> Transfer the lysate into the QIAprep spin column, being careful not to transfer any solid. Discard the microcentrifuge tube with the solid</li><br />
<li> Centrrifuge the collection tube at 14 000 rpm for 1 minute. DIscard whatever liquid flowed through the binding column into the collection tube</li><br />
<li> Add 750 &micro;L of Buffer PE with concentrated ethanol added to the column and spin at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube</li><br />
<li> Repeat Step 12 a second time with the same quantity of Wash Solution</li><br />
<li> Centrifuge the tube for 1 minute at 14 000 rpm to dry the column</li><br />
<li> Transfer the column to a new 1.5 &micro;L microcentrifuge tube</li><br />
<li> Add 50 &micro;L of double distilled water to the column and spin for 1 minute at 14 000 rpm</li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
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<br />
<br />
<a name="GE"></a><br />
<br /><br /> <h2 style="color:#0066CC">Gel extraction</h2><br />
<br /><br />
<p>This protocol is utilized in accordance to the manufacturer's protocol from Omega E.Z.N.A (EaZy Nucleic Acid Isolation)</p><br />
<br />
<ol><br />
<li> Place gel on the UV box</li><br />
<li> Carefully extract the fragment suspended in the gel>/li><br />
<li> Mass gel fragments </li><br />
<li> Place fragment into a 1.5 mL tube and add 4 &micro;L of H2O </li><br />
<li> Volume of water added to volume of gel is 200% however if fragment it small 1 mL of water will suffice </li><br />
<li> Remove H2O </li><br />
<li> Add equal amounts of H2O and Binding Buffer (XP2) to the gel</li><br />
<li> Incubate mixture at 55 degrees for 7 mins </li><br />
<li> Mix with vortex for 2 mins </li><br />
<li> Place in the HiBind DNA Mini Column in the 2 mL tube </li><br />
<li> Add 700 &micro;L at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Add 300 &micro;L Binding Buffer (XP2) into the HiBind DNA Mini Column and spin down at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Wash the column with 700 &micro;L of SPW buffer with added ethanol and spin down at 10,000xg for 1 min </li><br />
<li> Discard liquid </li><br />
<li> Wash the column with 700 &micro;L of SPW buffer again and spin down at 10,000xg for 1 min </li><br />
<li> Discard the liquid </li><br />
<li> Spin down the column at 13,000xg for 1 min to dry the column </li><br />
<li> Elute in 50 &micro;L of H2O and wait 1 min </li><br />
<li> Spin down the column at 13,000xg for 1 min to dry the column </li><br />
<li> Use a spectrophotometer to measure the concentration and the purity of your plasmid</li><br />
</ol><br />
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<br />
<br />
<br />
<a name="PP"></a><br />
<br /><br /><h2 style="color:#0066CC">PCR purification (Vaccum)</h2><br />
<br /><br />
<p>This protocol is utilized in accordance to the manufacturer's protocol from Qiagen PCR Vacuum Prep Kit </p><br />
<ol><br />
<li> Add PCR product</li><br />
<li> Distribute liquid evenly by evenly tapping</li><br />
<li> Vacuum for 15-20 mins to dry</li><br />
<li> Add 20 &micro;L H2O</li><br />
<li> Tap lightly against the table to redistribute the H2O on the surface of the well</li><br />
<li> Pipette contents and transfer to clean 1.5 mL tube</li><br />
</ol><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Project/ControlsTeam:Calgary/Project/Controls2010-10-27T09:31:32Z<p>Pauladamiak: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Testing our system</h1></span><br />
<br />
<p><br />
Once constructed, we needed a way to test the cytoplasm and periplasmic stress promoters in order to characterize them. We did this in three different ways.<br />
</p><br />
<ol><br />
<li>tetsing with known folding and misfolding proteins<br /></li><br />
<li>tetsing with NLPE, an outer membrane lipoprotein known to activate the cpx pathway<br /></li><br />
<li>tetsing with varying temperature conditions<br /></li><br />
</ol><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with known folding and misfolding proteins</h2><br />
<p><br />
We first needed to identify proteins that we know fold and don’t fold well inE. coili. For this we chose the maltose binding protein. This is a protein known to fold extremely well in the periplasm of E. Coli. MalE31, a mutant with two amino acid substitutions at postion 33 and 34, does not fold and is classified as a non-folder. MalE with the signal sequence removed, does not move in to the periplas, but remains in the cytoplasm where it folds extremely well. Male31 with the signal sequence removed, is a non folder in the cytoplasm. Thus we have four proteins coverning folding and non-folding in botht he periplas and the cytoplasm.<br />
</p><br />
<div style="width:400px; height:400px; border:1px solid black"><p>maltose binding chart place holder</div><br /><br />
<br />
<p>We received these genes from the Betton labs in France. We biobricked these parts, but before testing our stress reporters with them, we wanted to first test these parts to sohow that they work as expected.. To do this, we transformed them into strains of cells containing cpxR and degP promoters up stream of a lacZ rpeorter (Raivio labs). We would expect malE31, if it misfolded, to activate the cpxR and degP stress promoters, thsus providing a blue output from lacZ. MalE on the other hand would not misfod, and therefore would not activate these promoters, and we would not expect to see any lacZ activity. This allowed us to conlcude that malE and malE31 work the way that we expected them to. See results on our characterization page.</p><br />
<br />
<p><br />
Once malE and malE31 were shown to be functional, we then used them to test out the stress promoters. We did this by making competent cells containing our reporeter circuits. We then transofmed in exprressio constructs for our malE and mutant malE proteins. We then measured fluorescence output from our reporter constructs. See resuts for this on our characterization opage.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with NLPE</h2><br />
<p><br />
NLPE is an outer membrane lipoprotein that literature has shown actibates the cpX pathway. We transformed expression costructs for this protien (obtained from the Rvaio lab) into competent cells containing our cpxR reporter and looked for fluoresecnt output. Results for this experiment can be viewed on our characyerization page.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Testing with Varying Temperature Conditions</h2><br />
<p><br />
Finally we tested the cpxR promoter<br />
</p><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
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<p><br />
Once constructed, we needed a way to test the cytoplasm and periplasmic stress promoters in order to characterize them. We did this in three different ways.<br />
</p><br />
<ol><br />
<li>tetsing with known folding and misfolding proteins<br /></li><br />
<li>tetsing with NLPE, an outer membrane lipoprotein known to activate the cpx pathway<br /></li><br />
<li>tetsing with varying temperature conditions<br /></li><br />
</ol><br />
<br />
<br />
<h2 style="color:#0066CC">Testing with known folding and misfolding proteins</h2><br />
<p><br />
We first needed to identify proteins that we know fold and don’t fold well inE. coili. For this we chose the maltose binding protein. This is a protein known to fold extremely well in the periplasm of E. Coli. MalE31, a mutant with two amino acid substitutions at postion 33 and 34, does not fold and is classified as a non-folder. MalE with the signal sequence removed, does not move in to the periplas, but remains in the cytoplasm where it folds extremely well. Male31 with the signal sequence removed, is a non folder in the cytoplasm. Thus we have four proteins coverning folding and non-folding in botht he periplas and the cytoplasm.<br />
</p><br />
<div style="width:400px; height:400px; border:1px solid black"><p>maltose binding chart place holder</div><br /><br />
<br />
<p>We received these genes from the Betton labs in France. We biobricked these parts, but before testing our stress reporters with them, we wanted to first test these parts to sohow that they work as expected.. To do this, we transformed them into strains of cells containing cpxR and degP promoters up stream of a lacZ rpeorter (Raivio labs). We would expect malE31, if it misfolded, to activate the cpxR and degP stress promoters, thsus providing a blue output from lacZ. MalE on the other hand would not misfod, and therefore would not activate these promoters, and we would not expect to see any lacZ activity. This allowed us to conlcude that malE and malE31 work the way that we expected them to. See results on our characterization page.</p><br />
<br />
<p><br />
Once malE and malE31 were shown to be functional, we then used them to test out the stress promoters. We did this by making competent cells containing our reporeter circuits. We then transofmed in exprressio constructs for our malE and mutant malE proteins. We then measured fluorescence output from our reporter constructs. See resuts for this on our characterization opage.<br />
</p><br />
<br />
<h2 style="color:#0066CC">Testing with NLPE</h2><br />
<p><br />
NLPE is an outer membrane lipoprotein that literature has shown actibates the cpX pathway. We transformed expression costructs for this protien (obtained from the Rvaio lab) into competent cells containing our cpxR reporter and looked for fluoresecnt output. Results for this experiment can be viewed on our characyerization page.<br />
</p><br />
<br />
<h2 style="color:#0066CC">Testing with Varying Temperature Conditions</h2><br />
<p><br />
Finally we tested the cpxR promoter<br />
</p><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
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<span id="bodytitle"><h1>Periplasmic Stress Detectors</h1></span><br />
<br/><br />
<h2 style="color:#0066CC">What causes periplasmic stress?</h2><br />
<br />
<h2 style="color:#0066CC">Team Calgary circuits for periplasmic stress detector</h2><br />
<br />
<br />
<table><br />
<tr><td><br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxp-1.png"></img><br />
<br/><br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxr-1.png"></img><br />
<br/><br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/degp-1.png"></img><br />
</td><br />
<br />
<td><br />
These images illustrate the promoters of choice by team Calgary for detection of periplasmic stress. The promoters of choice include CpxP, CpxR and DegP promoters. </td></tr></table><br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat periplasmic stress?</h2><br />
<br />
<h3>CpxP</h3><br />
<br />
<h3>CpxR</h3><br />
<br />
<h3>DegP</h3><br />
<br />
<h2 style="color:#0066CC">Circuit usage and sensitivity</h2><br />
<br />
<br />
<p><br />
The circuit that will be constructed to detect protein misfolding will have the cpxP promoter. Two other similar circuits will be constructed to compare the activation of cpxP promoter compared to degP and cpxR promoter. All three promoters have the same function: activation of degP which degenerates misfolded proteins in the cell. As it can be seen in the graph below, the cpxP promoter is the most sensitive to the stress. <br />
The purpose of this circuit is to detect protein misfolding. For example, cpxP promoter becomes activated under several specific streses: elevation of pH and overexpression envelope proteins such as NlpE. Hence, if periplasmic misfolding occurs in the cell of an E.coli bacteria, the reporter gene, in this case the Red Fluroscent Protein (RFP), will be activated. Hence, the activation of the RFP will be the indication of a periplasmic protein misfolding.<br />
</p><br />
<br />
<br />
<br/><br />
<br />
<br />
</html>[[Image:DegP Cpx Project Figure.png|thumb|400px|left|DiGuiseppe, P.A., & Silhavy, T.J. (2003). Signal detection and target gene induction by the cpxra two-component system. Journal of Bacteriology, 185(8), 2436-2436.]]<html><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
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<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
<br />
<br />
<h2 style="color:#0066CC">How does protein misfolding occur?</h2><br />
<br />
<p>Protein misfolding can occur as a result of several factors. It can be<br />
due to overproduction of the protein in the cell, in which case the cell<br />
lacks resources such as chaperones to fold the protein fast enough. This<br />
can cause the proteins to misfold and form aggregate bodies. Proteins can<br />
also misfold due to mutations that occur in the coding region of the<br />
protein that can alter the amino acid sequence and thereby interrupting<br />
the native structure of the protein, causing it to misfold and be<br />
dysfunctional. Proteins can also misfold due to cellular stress such as a<br />
change in pH, temperature and change in media. Due to lack of optimal<br />
folding conditions proteins can form aggregate bodies and cause<br />
activation of heat shock systems, chaperone systems and proteolytic<br />
pathways which are involved in either refolding the proteins into their<br />
native form or degrading the aggregate bodies. Proteins can also misfold<br />
due to lack of localization. For example: if a periplasmic protein lacked<br />
a signal sequence it will misfold in the cytoplasm because the conditions<br />
are different in the two cellular compartments.</p><br />
<br />
<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress?</h2><br />
<br />
<p><br />
There are several heat shock pathways in E. coli which are actively<br />
transcribed in response to cellular stress. There are housekeeping genes called sigma factors that are responsible for maintaining homeostasis in the cell and helping with protein folding. Sigma32 is a factor that is crucial for maintaining and monitoring heat shock responses in the cytoplasm of <i>E. coli</i>. Sigma 32 and other house keeping factors act as transcription factors for small heat shock proteins (sHsps). sHsps consist of proteins such<br />
as ibpA, ibpB, DnaK, DnaJ, GroEL and GroES. Amongst these, IbpA (inclusion body binding proteins) and ibpB<br />
are two different proteins that are activated as a result of cytoplasmic<br />
stress response. IbpA and ibpB proteins are chaperones that are<br />
responsible for refolding aggregated bodies and inclusion bodies into<br />
their native conformation.</p><br />
<br />
<br />
<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
<br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
<br />
<td> The cytoplasmic stress detector has a fusion of sigma 32 activated heat shock promoter which allows a higher output compared to the ibpAB promoter and FxsA promoter </td><br />
<br />
</tr><br />
<br />
</table><br />
<br />
<h3>Rationale behind picking this promoter</h3><br />
<br />
<br />
<p><br />
In our cytoplasmic stress detector circuit, we decided to fuse two<br />
different promoter regions from two heat shock proteins, which are ibpAB<br />
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion<br />
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly<br />
higher output as a result of heat shock compared to the promoters<br />
individually. </p><br />
<br />
<table><br />
<tr><br />
<td><br />
<br />
<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=ibpAB-2.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-2.png" border="0" alt="ibpAB"></a> </td><br />
</tr><br />
</table><br />
<br />
<p><i>B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)</i></p><br />
<br />
<br />
<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
</p><br />
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<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Misfolding detection circuit overview</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p><br />
Protein misfolding can occur as a result of a variety of factors. Overproduction of proteins in the cell is a good example. When proteins are overproduced, the cell can become overwhelmed and lack the necessary resources such as chaperones in order to deal with the large amount of protein. Proteins can also misfold due to mutations that occur in the coding region of the protein that can alter the amino acid sequence thereby interrupting the native structure of the protein. This can cause it to misfold into a non-functional state. Proteins can also misfold due to cellular stress such as changes in pH, temperature and changes in media. Localization can also be an issue. If a periplasmic protein lacks a signal sequence for example, it could misfold in the cytoplasm because the conditions are different in the two cellular compartments.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">Why do we care?</h2><br />
<p><br />
Protein misfolding is an important topic in mnay regards. Many diseases, particularly neurodegenerative disorders such as Alzheimer’s Disease and __ result from misfolding proteins. The production recmonbaint proteins in prokaryoes such as E. Coli can also pose a problem. Non-native proteins are more susceptible to misfolding. This can compliacte many lab projectas such as the deisgn of protein drugs.<br />
</p><br />
<br /><br />
<br />
<h2 style="color:#0066CC">How does our system detect protein misfolding?</h2><br />
<h3>Current methods</h3><br />
<p><br />
GFP fusions are a method commonly used to detect protein misfolding. Targeted proteins can be fused to the C-Terminal of reporter genes such as GFP or Luciferase. If the target gene folds correctly, it would permit the reporter gene to also fold correctly, thus giving a measurable output. If the target gene was not able to fold however, the thought is that the reporter gene would not be able to fold correctly either, Arguments have been made however, that the fusion may affect the solubility of the target protein, thus resulting in an ineffective testing system. A more recent system has been the use of a split GFP system. Cabantous et al (2005) describe a system using two fractions of GFP. The smaller part is fused to the target protein. The small size of the fraction of GFP fused to the target protein is thought to not affect the solubility of the protein of interest. Nevertheless, many heterologous proteins often are not suitable for fusion with such reporters due to inaccessible C terminus of the target protein. <br />
</p><br />
<br />
<h3>Our System</h3><br />
<p><br />
Another method of protein misfolding detecton is thus to look at transcription levels of different heat shock promoters. By monitoring the activity levels of native stress promoters, you cab look more to the cell to report in its own stress levels. Because the reporter itself is decoupled from the stress, there is a minimized chance of the reporter having a stabilizing effect on the misfolding protein.Because transcription from these promoters is drastically increased during times of stress in the cell, these promoters, when coupled with different reporter genes such as GFP or lacZ, can be used as indicators of protein misfolding, as this is a stress for the cell. <br />
</p><br />
<br />
<h3>Our stress promoters</h3><br />
<p><br />
We chose four stress promoters to look at: three that monitor stress in periplasm of E Coli: <a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a>, and one that monitors stress in the cytoplasm of E. Coli: <a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a><br />
</p><br />
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>Transcription/Translation Circuit</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p><br />
Transcription and translation are essential processes for protein expression. Problems that arise during these processes could lead to improper protein formation. Issues that can occur include shortage in length, folding problems, low or no expression, etc. These issues are accentuated in synthetic biology as foreign genes are implemented into prokaryotes such as ''Escherichia coli''. The transcription translation detector circuit was developed in order to test whether or not a gene of interest is being correctly transcribed and translated.</p><br /><br />
<br />
<h3>How the Circuit Works</h3><br />
<div style="border:1px solid grey; width:500px; height:200px"><p> place ciruct image here </p> </div><br /><br />
<table><br />
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<img style="width:100px; height:100px" src="https://static.igem.org/mediawiki/2010/7/70/Random.png" title="random2" /><br /><br />
</td><br />
<td><br />
<p><br />
The gene of interest is fused to a mutant RFP. Downstream of this is GFP with its own ribosomal binding site. If trabnscription is occuring, the transcript would include the gene of interest, RFP as well as GFP. Because GFP has its own ribosomal biding site, it should be translated if transcription is happening.</p><br />
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<br/><br />
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<img style="width:100px; height:100px" src="https://static.igem.org/mediawiki/2010/7/70/Random.png" title="random2" /><br /><br />
</td><br />
<td><br />
<p><br />
If the gene of interest is also being translated, then RFP should also be translated because it is fused to the GOI.the RFP was specially selected from Dr. Lewenza’s lab. This RFP (nicknamed sRFP or special red fluorescent protein) can fold in the cytoplasm, periplasm and the cellular membrane.<br />
</p><br />
</td><br />
</tr><br />
</table><br /><br /><br />
<br />
<h2 style="color:#0066CC">Details</h2><br />
<h3>Assumptions:</h3><br />
<p><br />
For this circuit to work, there are several assumptions that must be made. The first of which is a result of a limitation within the design which is that sRFP will not affect the stability of the protein of interest. Both positives and negatives are not ideal because the circuit functions as an indicator any assistance could lead to false positives or vice versa. Second, AraC is the right promoter for this circuit. Although there are many benefits for using an arabinose inducible promoter, however evolutionary conditions have established optimal expression in natural promoters. Third is folding properties in the periplasm and cytoplasm (Lewenza, et al., 2006) had to be the same such that a sRFP in the cytoplasm will give the same absorbance as one in the periplasm. Fourth would be that the GOI does not contain a “rut” site (Rho utilization site) which would prematurely stop transcription using Rho dependent termination. Fifth would be that E.coli would be the most compatible cell available for protein expression. Much like the second assumption, genes are optimally expressed in its natural host. Transferring these genes into E.coli might decrease the efficiency of protein expression. These are considerations that must be made in order to ensure the success of this circuit towards its utilization within our testing kit. It is definitely more “artificial” compared to the other two mostly because it overrides the necessity for the natural systems within. However if all limitations are accounted for, this could be a very useful tool if coupled with our other systems. </p><br />
<br />
<p>Helpful tips with understanding the circuit: With the way the circuit is developed, a failure of transcription will lead to a failure of translation. Therefore it is impossible to see only red cells, but possible to see green cells. If a brownish color is expressed (a mixture of red and green), this is the best. Also if only green cells are noticed, then to definitively test whether or not there is something wrong with translation, a user must employ the other two circuits. Meaning positive in the folding circuits indicates the translation mechanism works however due to the design of attaching sRFP with the GOI, the GOI misfolding will affect the stability of sRFP.</p><br /><br /><br />
<br />
<h3>Problems that can arise during transcription / translation:</h3><br />
<p>There are numerous problems that can arise in the transcription and translation especially when trying to turn E.coli into a factory for foreign proteins. Each category of transcription and translation can be broken down into pre, during and post. Although some aspects between post-transcription and pre-translation are slightly grey, there are parts of it that are quite clear. For example the attachment of the 30S subunit from rRNA would be considered pre-translational but not post-transcriptional. This section describes some of the possible transcription/translation issues and the following responses by the system. </p><br /><br />
<br />
<h4 style="color:#003366">Transcription</h4><br />
<p><b>Pre-transcription</b></p><br />
<p><em>transcription factors</em></p><br />
<p>One of the main ideas of synthetic biology is the expression of proteins from foreign enzymes, for example GFP comes from Aequorea Victoria (Andersen, et al., 1998) . One of the considerations is whether or not foreign circuits have the corresponding transcriptions within E.coli. If these transcription factors have a profound effect on whether or not transcription can occur (Kleinert, et al., 2003) , then natural promoters might be hindered or lack the necessary transcription factors for expression. Therefore it is necessary to include an arabinose promoter (pBad/araC), a well characterized and working promoter in E.coli. (iGEM registry,2003) </p><br />
<br />
<p>If the problem of the foreign circuit lies in the promoter, the circuit can be used to detect this simply through inserting the RBS+GOI into the circuit and compare this with inserting the foreign promoter + RBS + GOI. If there is expression without the foreign promoter, and no expression with it, then there could be a repressor bounded to the operon of the circuit. If there is expression in both then a third circuit can be constructed with just the foreign promoter + RBS + GOI without the arabinose promoter. If there is no expression in the third, then the foreign promoter lacks the necessary transcription factors to operate in the host E.coli.</p> <br />
<br />
<p><em>promoter strength</em></p><br />
<p>This is not a problem with natural promoters however this is an issue faced by many synthetic biologist when matching a promoter with a GOI. More is not always better, over expression of protein could lead to higher amounts of aggregation and longer folding time.(Brock, 2010) Choosing the pBAD/araC promoter is beneficial because induction varies with arabinose concentrations. Therefore it is possible to use a 96 well plate with varying levels of arabinose to promote induction at various strengths. A plate reader can then be used to read absorbance levels to find the optimal amount of indicator expressed.</p><br /><br />
<br />
<p><b>Transcription</b></p><br />
<p><em>Repressor/amount of inducer</em></p> <br />
<p>The ratio of inducer to plasmid copy number would be a problem when trying to express a foreign protein in E.coli. Much like issue with transcription factors, the circuit was designed to include an arabinose promoter that way it is possible to control the concentration of the inducer arabinose. In that case there will be no shortage in the concentration of inducer because the promoter is well characterized meaning its induction is known.</p><br />
<br />
<p><em>Hair pin loop/rho dependent termination</em></p><br />
<p>The formation of premature hair pin loops and rho utilizations sites formed from within the gene are potential methods of premature stops to transcription. Hair pin loops are typically 7 to 20 amino acids long (Lewin, 2007) and ruts sites are 22-116 base pairs.(Banerjee, et al., 2007) The more likely of the two when forming an accidental termination site would be a hair pin loop. This relies on the palindrome formation with high concentrations of guanine and cysteine which results in a RNA pulling from the DNA. Our system would detect premature termination of RNA would result in no signal with our GFP signal.</p><br /><br />
<br />
<br />
<p><b>Post-transcription</b></p><br />
<p><em>mRNA shape degradation</em></p><br />
<p>Although transcription occurs, mRNA instability results in the degradation of the mRNA. The circuit would suggest that the GFP report was not expressed. Despite transcription occurring completely, the most logical approach would be to group this under issues with transcription, also because pre-translational steps have not occurred yet.</p><br /><br />
<br />
<h4 style="color:#003366">Translation</h4><br />
<p><b>Pre-translation</b></p><br />
<p>No current issues arise from this step.</p><br /><br />
<p><b>Translation</b></p><br />
<p><em>Multi codon usage</em></p> <br />
<p>When inserting foreign genes into E.coli, the ratios of tRNAs in E.coli in comparison to the foreign source can vary. Shortages in tRNA can lead to problems with rate and accuracy of translation. (Ran and Higgs, 2010) Kinetics is a factor of rate of protein formation, decreased concentrations of necessary tRNAs results in slower formation of proteins. Based on the research by Drummond and Wilke, lack of accuracy is caused by mistranslation causing higher amount of misfolding.(Drummond and Wilke, 2008) If the GOI’s multi codon usage disagrees with the host E.coli, there would be aggregation which will inhibit protein expression. The circuit can detect that there are problems with translation because sRFP would be form aggregate bodies with the protein of interest (POI). </p><br />
<br />
<br />
<p><em>Premature stop codon</em><p><br />
<p>Stop codons inhibit translation. The circuit would indicate the presence of a premature stop codon because the sRFP would not be translated therefore no signal would be present. </p><br />
<br />
<p><em>RBS compatibility</em></p><br />
<p>The ribosome binding site allows the attachment of the ribosome. Differences in ribosome strength could change the translation frequencies. This leaves room for protein misfolding. Because of the specificity of the RBS to the expression of the gene, as well as the potential of affecting the triple nucleotide site which could shift the reading frame. The circuit was designed in such a way that the user is capable of attaching their own RBS.</p><br />
<br />
<p><em>Copy number</em></p><br />
<p>Copy number refers to the number of plasmids that can exist within on E.coli cell.(iGEM Registry, 2009) Although this doesn’t change the rate of transcription (polymerase per second, PoPs) like promoter strength, the effects are similar. Increasing the concentration of slower folding proteins could result in aggregation due to exposed hydrophobic segments. . (Ran and Higgs, 2010) The circuit will detect this as an issue with translation as this could affect the protein.</p><br /><br />
<br />
<p><b>Post-translation</b></p><br />
<p><em>Lack of chaperones</em></p><br />
<p>The lack of essential chaperones could result in protein misfolding. E.coli may not have the necessary chaperones to correct the conformation of the POI. The formation of misfolded protein will cause the aggregation of pRFP, therefore indicating an error in translation.</p><br /><br /> <br />
<br />
<h2 style="color:#0066CC">Design of the Circuit</h2><br />
<p>pBad/araC Promoter- This promoter was chosen because it allow for variable strength without replacing the promoter (if the circuit had a promoter library). Because more isn’t always better, the user can customize optimal levels of promoter strength in protein expression. This part is also highly characterized (iGEM Registry, 2003).</p><br />
<br />
<p>Multiple Cloning Sites- We are using modified biobrick prefix and suffix. What this means is that these sites are not separating the biobrick parts from the sequences, rather they are located between the arabinose promoter and the sRFP.</p><br />
<p>ccdB- A place holder that was added for selection in addition to antibiotic selection. The circuit will contain a suicide ccdB gene as a placeholder for the GOI. If this is not removed, the cell which has this transformed plasmid will die. This will ensure that the only cells present on the plate will only express the genes intended to be there.</p> <br />
<p>RBS- We have decided not include a RBS within this sequence to allow customizability. Natural RBS are known to indicate optimal PoPs plus issues with this would indicate compatibility problems on the part of the RBS and GOI. This would also eliminate any issues regard reading frame shifts of the RBS to the GOI for those that are biobricking new parts.</p> <br />
<p>sRFP (special red fluorescent protein)- This is part of the translation portion of the circuit. This indicator was chosen because it can fold in the cytoplasm, periplasm and membranes.(Lewenza, et al., 2006) One of the limitations of this circuit is that the GOI must be fused to sRFP in order for translation detection to occur. This means additional time on the part of the user to rebiobrick the end portion of the GOI such that the stop codons are removed. Current studies by Lewenza, et al. reveals that RFP can be localized in the cytoplasm as well as the outer membrane.</p> <br />
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">Testing our system</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<div style="width:400px; height:400px; border:1px solid grey"><p> central dogma image</p></div><br /><br />
<br />
<h2 style="color:#0066CC">Sites of failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>transcription image here</p></div><br />
</td><br />
<td><br />
<p> transcription text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Tranlsation</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>translation image here</p></div><br />
</td><br />
<td><br />
<p> translation text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>Folding image here</p></div><br />
</td><br />
<td><br />
<p> folding text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Our circuit and conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of conventional methods</h3> <br />
<p>The most commonly used test for transcription and translation is the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These Thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of disadvantages of conventional methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of our circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2010-10-27T09:15:11Z<p>Pauladamiak: </p>
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<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<div style="width:400px; height:400px; border:1px solid grey"><p> central dogma image</p></div><br /><br />
<br />
<h2 style="color:#0066CC">Sites of failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>transcription image here</p></div><br />
</td><br />
<td><br />
<p> transcription text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Tranlsation</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>translation image here</p></div><br />
</td><br />
<td><br />
<p> translation text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>Folding image here</p></div><br />
</td><br />
<td><br />
<p> folding text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Our circuit and conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of conventional methods</h3> <br />
<p>The most commonly used test for transcription and translation is the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These Thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of disadvantages of conventional methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of our circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2010-10-27T09:14:43Z<p>Pauladamiak: </p>
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<div class="container"><br />
<br />
<br />
<div class="sidebar"><br />
<br />
<br />
<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<div style="width:400px; height:400px; border:1px solid grey"><p> central dogma image</p></div><br /><br />
<br />
<h2 style="color:#0066CC">Sites of failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>transcription image here</p></div><br />
</td><br />
<td><br />
<p> transcription text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Tranlsation</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>translation image here</p></div><br />
</td><br />
<td><br />
<p> translation text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>Folding image here</p></div><br />
</td><br />
<td><br />
<p> folding text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Comparison of our circuit and conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of conventional methods</h3> <br />
<p>The most commonly used test for transcription and translation is the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These Thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of disadvantages of conventional methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of our circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2010-10-27T09:13:40Z<p>Pauladamiak: </p>
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<div class="container"><br />
<br />
<br />
<div class="sidebar"><br />
<br />
<br />
<h1>Project Descriptions</h1><br />
<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<div style="width:400px; height:400px; border:1px solid grey"><p> central dogma image</p></div><br /><br />
<br />
<h2 style="color:#0066CC">Sites of failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>transcription image here</p></div><br />
</td><br />
<td><br />
<p> transcription text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Tranlsation</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>translation image here</p></div><br />
</td><br />
<td><br />
<p> translation text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>Folding image here</p></div><br />
</td><br />
<td><br />
<p> folding text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Advantages of our system over conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<h3>Overview of conventional methods</h3> <br />
<p>The most commonly used test for transcription and translation is the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These Thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<h3>Discussion of disadvantages of conventional methods</h3><br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<h3>Advantages of our circuit</h3> <br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/ProjectTeam:Calgary/Project2010-10-27T09:10:14Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
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</ul><br />
<br />
</div><br />
<br />
<div class="mainbody"><br />
<br />
<span id="bodytitle"><h1>The Project</h1></span><br />
<br />
<h2 style="color:#0066CC">Overview</h2><br />
<p>Many synthetic biology projects involve the expression of recombinant proteins in microorganisms such as E. coli. The problems encountered with many synthetic biology projects often involve problems with protein expression. It is often very difficult to recognize the problem and pinpoint where it lies. The goal of the University of Calgary 2010 iGEM team is to build a protein expression "troubleshooting kit". This kit will contain two systems with which target genes can be inserted. In the resulting cell growth, fluorescent protein production will be used to determine whether there is a problem with protein expression as well as indicate where the protein expression is failing.</p><br />
<br />
<br />
<p><br />
Protein expression happens in three steps: the transcription of the DNA to mRNA, the translation of mRNA into an amino acid sequence, and the folding of that amino acid sequence into a protein.</p><br />
<div style="width:400px; height:400px; border:1px solid grey"><p> central dogma image</p></div><br /><br />
<br />
<h2 style="color:#0066CC">Sites of failure</h2><br />
<p><br />
Protein expression can fail at any point along these three steps. Our system uses two circuits to detect at which step possible errors have occured. The first circuit has a fluorescent reporter that is produced when DNA is transcribed into mRNA and another that is produced when mRNA is translated into a functional protein. When both reporter proteins are expressed in the cell, it indicates both transcription and translation are successful. The second circuit involves reporter systems that are activated as a result of protein misfolding. Two native stress-activated promoters from E. coli were engineered upstream to fluorescent reporters that will respond to periplasmic and cytoplasmic protein misfolding. If the protein of interest misfolds in either area of the cell, one of the promoters will be activated and the corresponding fluorescence will be observed.<br />
</p><br />
<br />
<h3>Transcription</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>transcription image here</p></div><br />
</td><br />
<td><br />
<p> transcription text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<br />
<h3>Tranlsation</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>translation image here</p></div><br />
</td><br />
<td><br />
<p> translation text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h3>Folding</h3><br />
<table><br />
<tr><br />
<td><br />
<div style="width:200px; height:100px;"><p>Folding image here</p></div><br />
</td><br />
<td><br />
<p> folding text here</p><br />
</td><br />
</tr><br />
</table><br /><br />
<br />
<h2 style="color:#0066CC">Advantages of our system over conventional transcriptional and translational tests</h2><br />
<p>The problem faced by many researchers today is difficulty in locating which step of the protein expression process is malfunctioning. iGEM teams are additionally constrained by a deadline. Our Toolkit has made it possible to pinpoint the exact process in which errors are occurring (transcription, translation, cytoplasmic protein folding or periplasmic protein folding), over the period of a few days.</p><br />
<br />
<p>The most commonly used test for transcription and translation is the Northern and Western Blot respectively. Northern Blot can be used to detect transcription because it relies on the isolation of mRNA. mRNA is typically extracted from a sample using oligo (dT) – cellulose chromatography. Essentially, this method exploits the poly-A tail characteristic of mRNA. In living cells, there are three types of naturally occurring RNA: rRNA, tRNA and mRNA. mRNA has a segment of (~250) Adenine nucleotides on the 3’ end that enhances both the lifetime and translatability of mRNA. The sample can be ran through a column containing oligo dT or deoxyribose Thymine nucleotides. These Thymine nucleotides act as a sort of ‘primer’ such as primers in a PCR binding with the Poly-A tail. This ‘double stranded’ mRNA complex can be eluted out with a slight pH fluctuation. The isolated mRNA is then ran on a gel allowing it to segregate by size, and then blotted onto a nylon membrane. A positively charged nylon membrane is often more effective as the negatively charged nucleic acids have a higher affinity for it. The blotted membrane is then transferred to a transfer buffer containing RNA probes complimentary to the RNA sequence of interest. RNA is very unstable and is often degraded by factors such as high temperatures therefore the blotting process needs occur in the presence of formamide which helps lower the probe-RNA interaction temperature. Formamide is highly corrosive to the skin and an extreme teratogen. The membrane can then be examined under UV light, which will allow the RNA-probe complexes to fluoresce. This indicates that the DNA sequence of interest is being translated. </p><br />
<br />
<p>From this procedure, we can see that there are limitations and disadvantages of using Northern Blot. Firstly, it is an extremely selective procedure. Concentrations of buffers, solvents and probes need to be optimal for the reaction to occur. Additionally, primer and probe sequences need to be exact. The entire Northen Blot procedure can take up to 8 days, which can be big issue for iGEM teams with stringent deadlines. Another major limitation is due the unstability of RNA, if RNA samples are even slightly degraded, the quality of the data and the ability to quantitate expression are severely compromised. For example, even a single cleavage in 20% of 4 kb target molecules will decrease the returned signal by 20%. Thus, RNase-free reagents and techniques are essential. The obvious final disadvatage is that Northern requires the use of harmful chemicals that need to be handled carefully.</p><br />
<br />
<p>Our transcription/translation circuit has many advantages over the Norther Blot. For one, it does not require the user to handle RNA and therefore it eliminates concerns regarding mRNA degradation. Another advantage would be that it does not require the use of harmful and carcinogenic chemicals, as it is just a simple construction of two DNA sequences that can then be transformed into cells. Our system also eliminates the specificity of primers and probes by eliminating the use of them together.</p><br />
<br />
<p>Our project is broken up into three smaller sections. Click any of the links on the side to learn more about each individual section.</p><br />
</div><br />
<br />
</div><br />
<br />
</body><br />
</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Project/AchievementsTeam:Calgary/Project/Achievements2010-10-27T08:50:17Z<p>Pauladamiak: </p>
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
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<br />
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<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
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<br />
<br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
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</ul><br />
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<br />
<span id="bodytitle"><h1>Periplasmic Stress Detectors</h1></span><br />
<br/><br />
<table><br />
<pr><pd><br />
<br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxp-1.png"></img><br />
<br/><br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/cpxr-1.png"></img><br />
<br/><br />
<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/degp-1.png"></img><br />
</pd></pr><br />
<br />
<pr><pd><br />
<p><br />
The circuit that will be constructed to detect protein misfolding will have the cpxP promoter. Two other similar circuits will be constructed to compare the activation of cpxP promoter compared to degP and cpxR promoter. All three promoters have the same function: activation of degP which degenerates misfolded proteins in the cell. As it can be seen in the graph below, the cpxP promoter is the most sensitive to the stress. <br />
The purpose of this circuit is to detect protein misfolding. For example, cpxP promoter becomes activated under several specific streses: elevation of pH and overexpression envelope proteins such as NlpE. Hence, if periplasmic misfolding occurs in the cell of an E.coli bacteria, the reporter gene, in this case the Red Fluroscent Protein (RFP), will be activated. Hence, the activation of the RFP will be the indication of a periplasmic protein misfolding.<br />
</p><br />
</pd></pr></table><br />
<br/><br />
<br />
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</html>[[Image:DegP Cpx Project Figure.png|thumb|400px|left|DiGuiseppe, P.A., & Silhavy, T.J. (2003). Signal detection and target gene induction by the cpxra two-component system. Journal of Bacteriology, 185(8), 2436-2436.]]<html><br />
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</html></div>Pauladamiakhttp://2010.igem.org/Team:Calgary/Project/IbpABTeam:Calgary/Project/IbpAB2010-10-27T08:49:21Z<p>Pauladamiak: </p>
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<h1>Project Descriptions</h1><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Transcription">Transcription/Translation Reporter Circuit</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/misfolding_overview">Protein Misfolding Reporters</a><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/IbpAB">Cytoplasmic Stress Detectors</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/CpxP">Periplasmic Stress Detectors</a></li><br />
</ul><br />
</li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Controls">System Controls</a></li><br />
<li><a href="https://2010.igem.org/Team:Calgary/Project/Achievements">Achievements</a></li><br />
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<span id="bodytitle"><h1>Cytoplasmic Stress Detectors</h1></span><br />
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<h2 style="color:#0066CC">How does protein misfolding occur?</h2><br />
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<p>Protein misfolding can occur as a result of several factors. It can be<br />
due to overproduction of the protein in the cell, in which case the cell<br />
lacks resources such as chaperones to fold the protein fast enough. This<br />
can cause the proteins to misfold and form aggregate bodies. Proteins can<br />
also misfold due to mutations that occur in the coding region of the<br />
protein that can alter the amino acid sequence and thereby interrupting<br />
the native structure of the protein, causing it to misfold and be<br />
dysfunctional. Proteins can also misfold due to cellular stress such as a<br />
change in pH, temperature and change in media. Due to lack of optimal<br />
folding conditions proteins can form aggregate bodies and cause<br />
activation of heat shock systems, chaperone systems and proteolytic<br />
pathways which are involved in either refolding the proteins into their<br />
native form or degrading the aggregate bodies. Proteins can also misfold<br />
due to lack of localization. For example: if a periplasmic protein lacked<br />
a signal sequence it will misfold in the cytoplasm because the conditions<br />
are different in the two cellular compartments.</p><br />
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<h2 style="color:#0066CC">How does a native <i>E. coli</i> cell combat protein related stress?</h2><br />
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<p><br />
There are several heat shock pathways in E. coli which are actively<br />
transcribed in response to cellular stress. There are housekeeping genes called sigma factors that are responsible for maintaining homeostasis in the cell and helping with protein folding. Sigma32 is a factor that is crucial for maintaining and monitoring heat shock responses in the cytoplasm of <i>E. coli</i>. Sigma 32 and other house keeping factors act as transcription factors for small heat shock proteins (sHsps). sHsps consist of proteins such<br />
as ibpA, ibpB, DnaK, DnaJ, GroEL and GroES. Amongst these, IbpA (inclusion body binding proteins) and ibpB<br />
are two different proteins that are activated as a result of cytoplasmic<br />
stress response. IbpA and ibpB proteins are chaperones that are<br />
responsible for refolding aggregated bodies and inclusion bodies into<br />
their native conformation.</p><br />
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<h2 style="color:#0066CC">iGEM Calgary cytoplasmic stress detection circuit</h2><br />
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<img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpab-1.png"></img> </td><br />
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<td> The cytoplasmic stress detector has a fusion of sigma 32 activated heat shock promoter which allows a higher output compared to the ibpAB promoter and FxsA promoter </td><br />
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<h3>Rationale behind picking this promoter</h3><br />
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<p><br />
In our cytoplasmic stress detector circuit, we decided to fuse two<br />
different promoter regions from two heat shock proteins, which are ibpAB<br />
and fxsA. In a study done by Kraft et al, they demonstrate that a fusion<br />
of IbpAB/fxsA promoters combined along with T7 DNA has a significantly<br />
higher output as a result of heat shock compared to the promoters<br />
individually. </p><br />
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<a href="http://s872.photobucket.com/albums/ab287/iGEMCalgary_2010/?action=view&current=ibpAB-2.png" target="_blank"><img src="http://i872.photobucket.com/albums/ab287/iGEMCalgary_2010/ibpAB-2.png" border="0" alt="ibpAB"></a> </td><br />
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<p><i>B: MalE31 induction with IPTG; C: MalE31 induction and reporter reading with just ibpAB promoter; D: MalE31 induction and reporter reading with just fxsA promoter; E: MalE31 induction and reporter reading with ibpAB/FxsA fusion promoter (Kraft et al, 2006)</i></p><br />
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<h3> How are we utilizing this promoter?</h3><br />
<p><br />
This fusion promoter will be connected to the registry part I13504 which<br />
is RBS-GFP-B0015. The ibpAB/fxsA circuit will be activated in the presence<br />
of aggregation in the cell. We will be using MalE31 with a signal sequence<br />
deletion (MalE31&#8710;SS) which was designed by Betton et al. The native<br />
E. coli protein MalE generally exported into the periplasmic space but<br />
this mutated protein does not get exported to the periplasmic space due to<br />
the signal sequence deletion. Also Betton et al designed MalE31such that<br />
there are two amino acid changes in the protein and it misfolds. The<br />
MalE31&#8710;SS protein coding region will be used in order to induce<br />
cytoplasmic protein stress in E. coli. </p><br />
<p>Ideally, this misfolded<br />
MalE31&#8710;SS should activate the plasmid system containing<br />
ibpAB/fxsA-I13504 which will produce GFP alerting the researcher that<br />
their protein is not being expressed in the cell because it is misfolding<br />
and as a result getting degraded. Our circuit should also be activated<br />
much faster than the native stress system because the ibpAB/fxsA promoter<br />
is much more sensitive to the presence of aggregate bodies in the cell.<br />
The promoter also gives a much higher output compared to the promoters<br />
individually, which is the case in the E. coli genome which should allow<br />
us to detect the fluorescence level much faster.<br />
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