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| | <div id="corps2"> | | <div id="corps2"> |
| - | <h3>Stage 3 : Design of synthetic multifunctional enzymes</h3> | + | <h3>Stage 3 : Regulation</h3> |
| | <br> | | <br> |
| - | <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> | + | <p>The system of protein purification needs to be regulated to be fit. In fact, the cell machinery has limited energy sources to cope with the DNA replication, the proliferation, and simultaneously, the granule and the protein synthesis. We need to space out the synthesis of the different other elements.</p> |
| | <br /> | | <br /> |
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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> | + | <p>As described in the article (Banki et al., Protein Science, 2005), we planned to induce first the granule synthesis during 30 hours and then to start the protein synthesis, which can bind directly to the granules already shaped. The rate of synthesis will be better if we stop the granules synthesis at this time. So we need promoters which turn each other ON and OFF.</p> |
| | <br /> | | <br /> |
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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> | + | <p> We thought of two distinct systems in order to have an alternative if one of the promoters would not have been as good as expected. |
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| | + | </p> |
| | <br /><br /><br /> | | <br /><br /><br /> |
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| - | <h3>Theory</h3> | + | <h3></h3> |
| | <br /> | | <br /> |
| - | <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> | + | <p>First, we wanted to use the curli promoter in one of the system. We want to take advantage of the ability of this promoter to be ON at 28°C and OFF at higher temperatures. We looked for an iGEM promoter regulated by temperature, we chose a thermometer RNA. |
| - | <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>
| + | It is switched on above 32°C, allowing the transcription (BBa_K115017). |
| | + | </p> |
| | <br/> | | <br/> |
| | + | <p>When the two constructs, functional, are in the same cell, the culture is moved at 28°C so that the PHB granules are formed under the control of Curli promoter. Then, the culture is moved at 37°C allowing the synthesis of the target proteins, activated by the thermometer RNA. At 37°C, the Curli promoter is off and the PHB granules are not synthesized anymore. </p> |
| | <div style="text-align:center;"> | | <div style="text-align:center;"> |
| | <img class="image" src="http://upload.wikimedia.org/wikipedia/en/6/64/FASmodel2.jpg" | | <img class="image" src="http://upload.wikimedia.org/wikipedia/en/6/64/FASmodel2.jpg" |
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| | </div> | | </div> |
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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> | + | <p></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> | + | <p></p> |
| | <div style="text-align:center;"> | | <div style="text-align:center;"> |
| - | <img class="image1" src="https://static.igem.org/mediawiki/2010/3/32/Diapositive1.PNG" | + | <img class="image1" src="" |
| | alt="FAS model" /> | | alt="FAS model" /> |
| | </div> | | </div> |
| | <div style="text-align:center;"> | | <div style="text-align:center;"> |
| - | <img class="image2" src="https://static.igem.org/mediawiki/2010/6/62/Diapositive2.PNG" /> | + | <img class="image2" src="" /> |
| | </div> | | </div> |
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| | <div style="text-align:center;"> | | <div style="text-align:center;"> |
| - | <img class="image3" src="https://static.igem.org/mediawiki/2010/6/6d/Tableau_enzymes.png" /> | + | <img class="image3" src="" /> |
| | </div> | | </div> |
| | <br> | | <br> |
| - | <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> | + | <p> </p><br> |
| - | <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> | + | <p></p><br> |
| - | <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> | + | <p></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> | + | <p></p><br> |
| | | | |
| - | <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> | + | <p></p><br> |
| | | | |
| - | <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> | + | <p></p> <br> |
| | | | |
| | | | |
Stage 3 : Regulation
The system of protein purification needs to be regulated to be fit. In fact, the cell machinery has limited energy sources to cope with the DNA replication, the proliferation, and simultaneously, the granule and the protein synthesis. We need to space out the synthesis of the different other elements.
As described in the article (Banki et al., Protein Science, 2005), we planned to induce first the granule synthesis during 30 hours and then to start the protein synthesis, which can bind directly to the granules already shaped. The rate of synthesis will be better if we stop the granules synthesis at this time. So we need promoters which turn each other ON and OFF.
We thought of two distinct systems in order to have an alternative if one of the promoters would not have been as good as expected.
First, we wanted to use the curli promoter in one of the system. We want to take advantage of the ability of this promoter to be ON at 28°C and OFF at higher temperatures. We looked for an iGEM promoter regulated by temperature, we chose a thermometer RNA.
It is switched on above 32°C, allowing the transcription (BBa_K115017).
When the two constructs, functional, are in the same cell, the culture is moved at 28°C so that the PHB granules are formed under the control of Curli promoter. Then, the culture is moved at 37°C allowing the synthesis of the target proteins, activated by the thermometer RNA. At 37°C, the Curli promoter is off and the PHB granules are not synthesized anymore.
FAS I model, extracted from Wikipedia, visualization by Kosi Gramatikoff.
"
