Ljubljana/model

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<title>Company Name</title>
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       <h3><span>Model</span></h3>
       <h3><span>Model</span></h3>
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       <p class="p1"><span><b>Our aim was to build a theoretical model to show how the feedback loop made of T7 RNAP gene under control of its T7 promoter affects the behaviour of the system, particularly amplification. Initially, active T7 RNAP molecules are generated by any of three sources: split-ubiquitin formation, TEV protease reconstitution or cleavage off the membrane by HIV protease. In all the cases, T7 polymerase translocates into the nucleus, where it transcribes an effector gene, as well as T7 RNAP gene (self-amplification). The idea behind introducing such positive feedback loop was that the initial signal might be too low and could fade if not amplied to a sufficient level.</b></span></p>
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       <p class="p1"><span><b>We built a theoretical model to show how the positive feedback loop made of T7 RNA polymerase (T7 RNAP) gene under the control of its T7 promoter affects the behaviour of the system, particularly amplification. Initially, active T7 RNAP molecules are generated by any of the three sources: split-ubiquitin reconstitution and endogenour ubiquitin protease, TEV protease reconstitution or cleavage by the HIV protease. In all cases, T7 polymerase translocates into the nucleus, where it transcribes an effector gene, as well as T7 RNAP gene (self-amplification). The idea behind introducing such a positive feedback loop was that the initial signal might be too low and could fade if not amplied to a sufficient level.</b></span></p>
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<div id="products">
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       <h3><span>Model in Cell Designer</span></h3>
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       <h3><span>Model built in Cell Designer</span></h3>
       <p class="p1"><span>
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<img border="0" src="https://static.igem.org/mediawiki/2007/6/62/Model_low.jpg" width="650" height="488">
<img border="0" src="https://static.igem.org/mediawiki/2007/6/62/Model_low.jpg" width="650" height="488">
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Figure 1: Comprehensive view of the engineered pathways. Split ubiquitin pathway is shown on the lower half and HIV protease on the upper half. HIV protease synthesis pathway after infection is simplified in this model.
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<small><b>Figure 1:</b> Comprehensive view of the engineered pathway. Split ubiquitin pathway is shown at the lower half and HIV protease at the upper half. HIV protease synthesis pathway after the infection is simplified in this model.</small>
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HIV (human immunodeficiency virus) is a retrovirus, Its genome is composed of two single stranded RNA molecules. It has a gag/pol/env organization; gag genes (group specific antigen) code for structural proteins, env for proteins that build viral envelope, while pol genes are responsible for viral reproduction (they contain genes for reverse transcriptase, integrase and HIV protease).<br><br>
 
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HIV envelope consists of lipids and viral glycoproteins gp120 and gp41, which are crucial for binding of HIV to the host cell membrane and for entering into the cell. Inserted into the lipid bilayer are also other glycoproteins that guarantee firmness and protective function of the viral envelope. Gp120 binds to receptors (CD4) on the host cell surface, but additional co-receptors like chemokine receptors (CCR5, CXCR4) are also required for successful entry of HIV. Mutations in co-receptor genes can cause immunity – if HIV cannot enter host cells, HIV infection is prevented, and AIDS cannot develop.<br><br>
 
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The characteristic retroviral enzyme is reverse transcriptase, which transcripts viral RNA into DNA. Only DNA can integrate into host cell genome – this is the crucial step in expressing viral proteins that are needed for assembly of new viral particles. Viral gag and gag/pol genes are expressed as polyprotein; until this polyprotein is cut into functional units, it exerts no biological function. Polyprotein clipping is done by HIV protease. The resulting polyprotein fragments represent functional enzymes and structural proteins.<br><br>
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<div align="justify" id="support">
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      <h3><span>Signal Amplification</span></h3>
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      <p class="p1">
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Transcription of viral RNA into DNA and processing of the viral polyprotein are two most important steps in HIV replication cycle. These are thus obvious targets for HIV therapeutics. Inhibitors of reverse transcriptase and HIV protease are currently used to treat acute HIV infection.</a></span></p><br>
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Plots of the amount of the active T7 RNAP (red line) and effector (blue line) versus time. The infection begins at time 0. Parameters are selected arbitrarily and the simulation should be used to evaluate the behavior of the system under different gene ratios:<br><br>
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<a href="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg">
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<img border="0" src="https://static.igem.org/mediawiki/2007/4/47/SLOmodel2a.jpg" width="313" height="211">
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</a><br>
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<small><b>Figure 2a)</b> Simulation with 2 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small>
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</o:p></p>
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      <h3><span>Current Disease Treatment</span></h3>
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      <p class="p1">One of the first AIDS therapeutics were nucleotide or nucleoside analogues (NRTI – nucleoside-analogue reverse transcriptase inhibitors) – pseudosubstrates, that are during reverse transcription integrated into viral DNA instead of nucleosides and thus block the transcription. These drugs were superseded by non-nucleoside inhibitors (NNRTI) that could inhibit reverse transcriptase by binding into the alosteric site of the enzyme. The third type of drugs is a family of HIV protease inhibitors. In most cases specific inhibitors are very similar to protease substrates - the only difference is that because they cannot be cut, they block the active site by remaining bound into it.<br><br>
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Weakness of all these therapeutics is that they are very sensitive to HIV mutations – HIV can easily mutate and thus become drug resistant. A combination of drugs is used to minimize HIV's potential to develop resistance to each individual drug in the mixture. Some of the drugs induce mutations that have negative effect on the virulence and such drugs can be used in spite of developed resistance.<br><br>
 
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We still do not have a cure for AIDS that would be insensitive to HIV mutations. Our project presents new ways of potential AIDS therapeutics. Our approaches can be considered independent of HIV mutations. We have set up a few of biological ambushes; if HIV manages to avoid them, we presume that it would not be able to infect the cell anyway.</p>
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<a href="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg">
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<img border="0" src="https://static.igem.org/mediawiki/2007/4/43/SLOmodel2b.jpg" width="313" height="211">
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</a><br>
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<small><b>Figure 2b)</b> Simulation with 50 copies of the effector gene and no T7 RNAP genes present in the nucleus.</small>
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<a href="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg">
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<img border="0" src="https://static.igem.org/mediawiki/2007/c/c1/SLOmodel2c.jpg" width="313" height="211">
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</a><br>
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<small><b>Figure 2c)</b> simulation with 2 copies of the effector gene and 2 copies of T7 RNAP genes present in the nucleus (the red line is hidden under the green line because of the same transcription and translation kinetics parameters).</small>
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      <h3><span>Classes of Antiretroviral Drugs</span></h3>
 
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      <p class="p1">Antiretroviral drugs are mostly inhibitors of different stages in HIV life cycle. They are targeted at different enzymes or events that are typical for HIV infection – entry of the virus into the cell, reverse transcription, polyprotein cleavage... and are divided into seven main classes (REFERENCA!):<br>
 
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<a href="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg">
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<img border="0" src="https://static.igem.org/mediawiki/2007/e/e0/SLOmodel2d.jpg" width="313" height="211">
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</a><br>
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<small><b>Figure 2d)</b> simulation with 50 copies of the effector and 2 copies of T7 RNAP genes present in nucleus.</small>
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<a href="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg">
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<img border="0" src="https://static.igem.org/mediawiki/2007/6/65/SLOmodel2e.jpg" width="313" height="211">
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<small><b>Figure 2e)</b> simulation with 2 copies of the effector and 50 copies of T7 RNAP genes present in nucleus.</small>
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      <h3><span>Development</span></h3>
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      <p class="p1">Lorem ipsum dolor sit amet, <a href="#">consetetur sadipscing elitr, sed diam nonumy</a> eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. </p>
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As expected, in case <b>(a)</b>, where no additional amplification was designed, the amount of the effector increases linearily with time. With more copies of the effector gene present, the slope increases <b>(b)</b>. In case <b>(c)</b> with amplification through T7 RNAP, we were expecting the effector amount to grow exponentially. However, contrary to simple reasoning, this is not the case unless the number of effector gene copies is much larger than the number of T7 RNAP gene copies. This can be explained by a limiting rate of transcription; the maximal transcription rate is determined by how many polymerase molecules can bind to the promoter in a certain span of time. Therefore, if the number of polymerase molecules is already high, the system becomes saturated very soon and any further increase of polymerase concentration does not increase expression rate, so no exponential growth  of effector concentration occurs. However, if we increase the number of effector gene copies <b>(d)</b>, the system does not readily become saturated and we observe an exponential growth of effector concentration. After a certain time, the number of polymerase molecules increases, and effector genes become saturated. This again results in a linear growth of effector concentration.<br>
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<br>
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The model clearly showed that in order to successfully amplify the initiatial signal, it is optimal to use a much larger number of effector genes than of T7 RNAP genes.<br>
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</a></span></p><br>
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     <div id="footer">______________________________________<br />
     <div id="footer">______________________________________<br />
        
        
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<a href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>
|<a href="https://2007.igem.org/Ljubljana">Home</a>|
|<a href="https://2007.igem.org/Ljubljana">Home</a>|
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<a href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>
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<a href="https://2007.igem.org/Ljubljana/results">Results</a>
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<a href="https://2007.igem.org/Ljubljana">
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<img border="0" src="https://static.igem.org/mediawiki/2007/c/c6/BlurMetalHome.gif" width="34" height="34">
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         <h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3>
         <h3 class="menu"><span><a class="two" href="https://2007.igem.org/Ljubljana/AIDSplague">AIDS - Today's Plague</a></span></h3>
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          <li><a class="one" href="https://2007.igem.org/Ljubljana/AIDSplague">Epidemics</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/HIVbacground">HIV-1 Infection Background</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/Currenttreatment">Current Disease Treatment</a>&nbsp; </li>
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         <h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a></span></h3>
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         <h3 class="links"><span><a class="two" href="https://2007.igem.org/Ljubljana/Strategy">Project</a></span></h3>
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          <li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li>
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        <li><a class="one" href="https://2007.igem.org/Ljubljana/Strategy">Strategy</a>&nbsp; </li> 
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<li><a class="one" href="https://2007.igem.org/Ljubljana/implementation">Implementation</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li>
           <li><a class="one" href="https://2007.igem.org/Ljubljana/model">Model</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/achievements">Achievements</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Achievements</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/prospects">Prospects</a>&nbsp; </li>
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           <li><a class="one" href="https://2007.igem.org/Ljubljana/summary">Prospects</a>&nbsp; </li>
            
            
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<div id="ldiscussion">
<div id="ldiscussion">
         <h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3>
         <h3 class="discussion"><span><a class="two" href="https://2007.igem.org/Ljubljana/team">Team</a></span></h3>
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<li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Terms & References</a>&nbsp; </li>
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<div id="ldiscussion">
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<ul><li><a class="one" href="https://2007.igem.org/Ljubljana/glossary">Glossary & References</a>&nbsp; </li>
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li>
<li><a class="one" href="https://2007.igem.org/Ljubljana/acknowledgements">Acknowledgements</a>&nbsp; </li>
         </ul>
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Latest revision as of 18:47, 23 November 2007

Company Name

Model

We built a theoretical model to show how the positive feedback loop made of T7 RNA polymerase (T7 RNAP) gene under the control of its T7 promoter affects the behaviour of the system, particularly amplification. Initially, active T7 RNAP molecules are generated by any of the three sources: split-ubiquitin reconstitution and endogenour ubiquitin protease, TEV protease reconstitution or cleavage by the HIV protease. In all cases, T7 polymerase translocates into the nucleus, where it transcribes an effector gene, as well as T7 RNAP gene (self-amplification). The idea behind introducing such a positive feedback loop was that the initial signal might be too low and could fade if not amplied to a sufficient level.

For modelling interactions and enzymatic reactions, Michaelis-Menten kinetics for one or two substrates was used.

Model built in Cell Designer


Figure 1: Comprehensive view of the engineered pathway. Split ubiquitin pathway is shown at the lower half and HIV protease at the upper half. HIV protease synthesis pathway after the infection is simplified in this model.


Signal Amplification

Plots of the amount of the active T7 RNAP (red line) and effector (blue line) versus time. The infection begins at time 0. Parameters are selected arbitrarily and the simulation should be used to evaluate the behavior of the system under different gene ratios:


Figure 2a) Simulation with 2 copies of the effector gene and no T7 RNAP genes present in the nucleus.


Figure 2b) Simulation with 50 copies of the effector gene and no T7 RNAP genes present in the nucleus.


Figure 2c) simulation with 2 copies of the effector gene and 2 copies of T7 RNAP genes present in the nucleus (the red line is hidden under the green line because of the same transcription and translation kinetics parameters).


Figure 2d) simulation with 50 copies of the effector and 2 copies of T7 RNAP genes present in nucleus.


Figure 2e) simulation with 2 copies of the effector and 50 copies of T7 RNAP genes present in nucleus.


As expected, in case (a), where no additional amplification was designed, the amount of the effector increases linearily with time. With more copies of the effector gene present, the slope increases (b). In case (c) with amplification through T7 RNAP, we were expecting the effector amount to grow exponentially. However, contrary to simple reasoning, this is not the case unless the number of effector gene copies is much larger than the number of T7 RNAP gene copies. This can be explained by a limiting rate of transcription; the maximal transcription rate is determined by how many polymerase molecules can bind to the promoter in a certain span of time. Therefore, if the number of polymerase molecules is already high, the system becomes saturated very soon and any further increase of polymerase concentration does not increase expression rate, so no exponential growth of effector concentration occurs. However, if we increase the number of effector gene copies (d), the system does not readily become saturated and we observe an exponential growth of effector concentration. After a certain time, the number of polymerase molecules increases, and effector genes become saturated. This again results in a linear growth of effector concentration.

The model clearly showed that in order to successfully amplify the initiatial signal, it is optimal to use a much larger number of effector genes than of T7 RNAP genes.