Team:INSA-Lyon/Project/Stage3/Theory

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<h2>Regulation</h2>
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<br>
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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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<br />
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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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  <ul>
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<li><a href="/Team:INSA-Lyon/Project" class="blue"> > Droppy Project</a></li>
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    <li name="28"  
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<li><a href="/Team:INSA-Lyon/Project/Stage1/Theory" class="cteal"> > Stage 1</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage2/Theory" class="slateb"> > Stage 2</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Theory" class="yellow"> > Stage 3</a></li>
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    <a class="brn"> > At 28°C</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Theory" class="green"> > Theory</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Strategy" class="brn"> > Strategy</a></li>
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<li><a href="/Team:INSA-Lyon/Project/Stage3/Results" class="blue"> > Results</a></li>
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    <a class="orange"> > At 37°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/Modeling" class="green"> > Modelling</a></li>
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<IMG id="image" NAME="imagelegend" SRC="http://lh6.ggpht.com/_Uc3bmii-yi0/TMh7jxvKr0I/AAAAAAAAApI/8wc5Y0fiVhU/thReguLegend.PNG" alt="Theory Regulation Legend"/>
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<h3>Stage 3 : Design of synthetic multifunctional enzymes</h3>
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<p style="font-size:0.9em; text-indent:0px; text-align:center;"><em></em></p>
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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 multifunctional enzyme. </p>
 
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<h3>Theory</h3>
 
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<p>At the very beginning of our work, we analyzed the literature on storage lipid synthesis and realized that Evolution had performed synthetic biology long before us. </p>
 
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<p>In bacteria, such as E. coli or B. subtilis, and plants, the metabolic reactions leading to fatty acid synthesis are catalized by a collection of separate, "classical", monofunctional enzymes. However, in animals, the different enzymes are integrated into a single multifunctional protein in which substrates are handed from one functional domain to the next. In humans for instance, a 2511-residue polypeptide consisting of 7 domains contains all the catalytic components required to perform the 37 sequential reactions leading to the synthesis of palmitic acid from acetyl- and malonyl-CoA.</p>
 
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<div style="text-align:center;">
 
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<img class="image" src="http://upload.wikimedia.org/wikipedia/en/6/64/FASmodel2.jpg"
 
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alt="FAS model" />
 
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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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<p>This organization could be a result of the need of a highly precise spatial disposition of the components, as a consequence of one of the major challenges that any cell has to face : channeling large amounts of hydrophobic substances through an aqueous environment to membranes or storage granules. The great majority of microbes are able to synthesize lipid granules but, to our knowledge, these structure were never observed in E. coli before cloning of the genes responsible for the production of poly-hydroxy-butyrate. Considering this cryptic property of E. coli, we hypothesized that any cell possessed the capacity to produce the molecular structures needed to channel lipids to storage granules. Thus, we began to investigate these molecular structures and were particularly interested in understanding how discrete monofunctional enzymes could organize into one single multifunctional enzyme through evolution. In order to try to shed light on the evolution processes responsible for this molecular organization, and maybe find new ways to engineer storage lipid synthesis, we started an enthusiastic exploratory tour of genomic data bases, keeping in mind the famous Carl Woese's sentence "The cell is basically an historical document".</p> <br>
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<h3><font color="purple">Control under Arabinose</font></h3><br>
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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.  
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</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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<p>Fatty acid synthesis is an iterative process. At each cycle of four reactions, two atoms of carbon are incorporated into the growing chain. The enzymes catalizing these four reactions are ketoacyl-ACP reductase (KR), hydroxyacyl-ACP dehydrase (DH), enoyl-ACP reductase (ER), and ketoacyl-ACP synthase (KS). During the whole process, the fatty acid chain remains attached to a specific protein, the acyl carrier protein (ACP), which hands the molecule from one enzyme to the next. At the beginning of the process, the first acyl group is transferred from acetyl-CoA to ACP by an acyltransferase (AC). At the end of the process, termination of chain elongation occurs by removing the fatty acid from ACP, often by a transesterification to glycerol catalyzed by a thioesterase (TE).</p>
 
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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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<p>In E. coli, these six enzymes and ACP are encoded by seven distinct genes. In fungi and animals, they are integrated into a single protein, encoded by a single gene. So, from a technological point of view, the question is : what is the most efficient organization ? A set of discrete monofunctional enzymes or a single multifunctional protein ? A big project emerged from this question : to construct an E. coli strain producing its fatty acids with either its classical enzymes or a synthetic multifunctional peptide. This project involved 1) the conditional inactivation of the E. coli chromosomal gene encoding ACP (this objective could be reached by replacing its natural promoter by a temperature-dependent promoter), and 2) the construction of a gene containing the information for the seven activities. </p><br>
 
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<p>We were not able to reach these objectives in the frame of iGEM 2010 but we made progress toward the design of the synthetic gene.</p><br>
 
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<p>An important theoretical obstacle was the classical problem of expression of eukaryotic proteins in bacteria. Post-translational modifications could be needed, and some factors involved in the development of the appropriate structure could not be present in the E. coli cells. This obstacle was obviated when we discovered in the literature and the databases that multifunctional enzymes, with high similarities with the eukaryotic enzymes, still existed in bacteria.</p><br>
 
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<p>Several species of Actinomycetes, such as Mycobacterium tuberculosis, produce multifunctional enzymes involved in fatty acid synthesis. The organization of the domains of these bacterial enzymes is very similar to the organization of animal and fungal enzymes. The genes encoding these enzymes could therefore be cloned in E. coli, and their sequences may be used as models to design new bacterial enzymes.</p><br>
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<p>A comparison between the amino-acid sequences of the E. coli monofunctional enzymes and the corresponding domains of the multifunctional peptides revealed low similarities. An important part of a future project (iGEM 2011 ?) could be to replace the Actinomycete sequence of each domain by the sequence of the corresponding E. coli enzyme.</p><br>
 
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<p>Fatty acid synthesis is an iterative process. At each cycle of four reactions, two atoms of carbon are incorporated into the growing chain. The enzymes catalizing these four reactions are ketoacyl-ACP reductase (KR), hydroxyacyl-ACP dehydrase (DH), enoyl-ACP reductase (ER), and ketoacyl-ACP synthase (KS). During the whole process, the fatty acid chain remains attached to a specific protein, the acyl carrier protein (ACP), which hands the molecule from one enzyme to the next. At the beginning of the process, the first acyl group is transferred from acetyl-CoA to ACP by an acyltransferase (AC). At the end of the process, termination of chain elongation occurs by removing the fatty acid from ACP, often by a transesterification to glycerol catalyzed by a thioesterase (TE).</p> <br>
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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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