Team:INSA-Lyon/Project/Stage3/Theory

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<li><a href="/Team:INSA-Lyon/Project" class="blue"> > Droppy Project</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage1" class="cteal"> > Stage 1</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage2" class="slateb"> > Stage 2</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3" class="yellow"> > Stage 3</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Theory" class="green"> > Theory</a></li>
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<h2>Regulation</h2>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy" class="brn"> > Strategy</a></li>
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<br>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Results" class="blue"> > Results</a></li>
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<p>The system of protein purification needs to be regulated to be completely operational. In fact, the cell machinery has limited energy sources to cope with the DNA replication, proliferation, and simultaneously, the granules and proteins synthesis. We need to space out the synthesis of the different other stages.</p>
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<p>As described by Banki et al.(Protein Science, 2005), proteins can bind directly to the granules already shaped. Moreover the protein synthesis rate will be higher if the granules synthesis is stopped. Thus we planned to induce the granule synthesis during 30 hours before turning it off and starting the protein synthesis. So we needed promoters which turn each other ON and OFF. We designed two distinct systems in order to have an alternative if one of them didn't work as expected.
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<h3><font color="purple">Thermoregulation</font></h3><br>
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<p>First, we wanted to take advantage of our curli promoter which has the ability to be switched ON at 28°C and OFF at higher temperatures. You can read <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage3/Strategy/Theorycurli"> the theory </a> concerning curli and ompR to understand how this promoter and its regulators work.<br/></p>
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<p>
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Then, we looked for an iGEM promoter also regulated by temperature, we chose a thermometer RNA. 
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It is switched ON above 32°C, allowing the transcription (<a href="http://partsregistry.org/Part:BBa_K115017"> BBa_K115017</a>).
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<p>When both functional constructs are in the same cell, the culture is moved to 28°C so the PHB granules are synthesized under the control of Curli promoter. Then, the culture is moved to 37°C allowing the synthesis of the target proteins, activated by the thermometer RNA. At 37°C, the Curli promoter is turned OFF and the PHB granules are not synthesized anymore.</p>
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    <a class="brn"> > At 28°C</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Future_direction" class="coral"> > Future Direction</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Notebook" class="blue"> > Notebook</a></li>
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<li><a href="/Team:INSA-Lyon/Project/References" class="orange"> > References</a></li>
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    <a class="orange"> > At 37°C</a></li>
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<h3>Stage 3 : Evolution</h3>
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<p>Evolution has been naturally performing synthetic biology for the last thousands years without our knowledge. Evolution combined with mutation and environmental changes has designed and constructed new biological functions and systems not found so far in nature.</p>
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<p>This project aims to study how this happened in nature and to use this knowledge to engineer a more complex structure into a chassis that did not possess it.</p>
 
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<p> We were particularly interested in studying how discrete monofuntional enzymes got organised into one single multifunctional enzyme through evolution. Projects 1 and 2 are indeed dealing with the polyhydroxyalkanoate (pha) ABC operon gene which codes for 3 distinct enzymes. It would be the final goal of our global project to increase this lipid production by designing one optimized mutlifunctional enzyme. </p>
 
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<h3>Theory</h3>
 
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<p>We have studied the multi-enzyme Type I Fatty Acid Synthase (FAS I) as a model. This enzyme is indeed peculiar since it is able to catalyze 6 reactions, as shown on the drawing below, extracted from Wikipedia, visualization by Kosi Gramatikoff. This multifunctional polypeptide is not a single enzyme but can be visualized as a hand comprising functional domains and passing the substrates to one another. This 270 kDa heavy chain is approximately 2000 amino acids long and is present amongst mammals and fungi. This type of FAS has been characterized as being Type I (FAS I), in comparison with Type II Fatty Acid Synthase system (FAS II) which use discrete monofunctional enzymes for fatty acid synthesis. </p>
 
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<p style="font-size:0.9em; text-indent:0px; text-align:center;"><em>FAS I model, extracted from Wikipedia, visualization by Kosi Gramatikoff.</em></p>
 
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<h3><font color="purple">Control under Arabinose</font></h3><br>
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<p>The organization of those FAS, integrated in FAS I, discrete in FAS II, differ from one another but their mechanisms of elongation and reduction are quite alike. Each separated FAS II enzymes can be associated to its equivalent domain in FAS I.</p>
 
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<p>The evolutionary history of fatty acid synthases is therefore a tremendous source of information which will enrich our understanding of why and how FAS I and FAS II have co-evolved.
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<p>In an other way, we wanted to use the combination of the promoter pBad/araC and the promoter LuxR/cI.</p> <br>
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<p>Without arabinose, LuxR and LuxI proteins are synthesized constitutively. LuxI is involved in the synthesis of HomoSerine Lactone (HSL). The fixation of HSL in LuxR protein causes a conformational change of the protein. In this conformation, LuxR protein can interact with the promoter and activates it. This leads to PHB synthesis and when PHB molecules are accumulated enough, they organize themselves in granules.
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The promoter Pbad/araC, without arabinose, is in a off state.  
</p>
</p>
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<p>When arabinose is added to the medium, it interacts with Pbad/araC and induces the synthesis of cI protein and phasin-intein construction. cI protein negatively regulates the promoter LuxR/HSL and stops PHB synthesis. Its effect is more efficient than the effect of the LuxR/HSL complex. Therefore, the promoter regulated by LuxR/HSL is totally turned off. During this step, phasin and intein proteins are synthesized and get into PHB granules.<br><br> </p><br>
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<div style="text-align:center;">
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<img src="http://lh3.ggpht.com/_Uc3bmii-yi0/TMh-oPnKpUI/AAAAAAAAApc/2DNV5QBTPPs/avecetsansArabinose.PNG" alt="With and without Arabinose" title="With and without Arabinose"/>
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When the chain is 16 carbon atoms long, a last step is performed where the Acyl Carrier Protein is removed:
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<p>FAS I enzyme is therefore a multifunctional protein catalyzing the reactions of Malonyl and Acetyl Transferase, ß Ketoacyl-ACP Synthase, ß Ketoacyl-ACP Reductase, 3 Hydroxyacyl-ACP Dehydrase, Enoyl Reductase and Thioesterase, as refered in the table below with their corresponding EC_numbers.
 
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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Latest revision as of 19:34, 27 October 2010




Regulation


The system of protein purification needs to be regulated to be completely operational. In fact, the cell machinery has limited energy sources to cope with the DNA replication, proliferation, and simultaneously, the granules and proteins synthesis. We need to space out the synthesis of the different other stages.


As described by Banki et al.(Protein Science, 2005), proteins can bind directly to the granules already shaped. Moreover the protein synthesis rate will be higher if the granules synthesis is stopped. Thus we planned to induce the granule synthesis during 30 hours before turning it off and starting the protein synthesis. So we needed promoters which turn each other ON and OFF. We designed two distinct systems in order to have an alternative if one of them didn't work as expected.



Thermoregulation



First, we wanted to take advantage of our curli promoter which has the ability to be switched ON at 28°C and OFF at higher temperatures. You can read the theory concerning curli and ompR to understand how this promoter and its regulators work.

Then, we looked for an iGEM promoter also regulated by temperature, we chose a thermometer RNA. It is switched ON above 32°C, allowing the transcription ( BBa_K115017).


When both functional constructs are in the same cell, the culture is moved to 28°C so the PHB granules are synthesized under the control of Curli promoter. Then, the culture is moved to 37°C allowing the synthesis of the target proteins, activated by the thermometer RNA. At 37°C, the Curli promoter is turned OFF and the PHB granules are not synthesized anymore.

Theory Regulation Theory Regulation Legend





Control under Arabinose



In an other way, we wanted to use the combination of the promoter pBad/araC and the promoter LuxR/cI.


Without arabinose, LuxR and LuxI proteins are synthesized constitutively. LuxI is involved in the synthesis of HomoSerine Lactone (HSL). The fixation of HSL in LuxR protein causes a conformational change of the protein. In this conformation, LuxR protein can interact with the promoter and activates it. This leads to PHB synthesis and when PHB molecules are accumulated enough, they organize themselves in granules. The promoter Pbad/araC, without arabinose, is in a off state.

When arabinose is added to the medium, it interacts with Pbad/araC and induces the synthesis of cI protein and phasin-intein construction. cI protein negatively regulates the promoter LuxR/HSL and stops PHB synthesis. Its effect is more efficient than the effect of the LuxR/HSL complex. Therefore, the promoter regulated by LuxR/HSL is totally turned off. During this step, phasin and intein proteins are synthesized and get into PHB granules.


With and without Arabinose

Top of Page

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