Team:INSA-Lyon/Project

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<h2>The <em>Droppy project</em> !</h2>
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<li><a href="/Team:INSA-Lyon/Project/Theory" class="cteal"> > Theory</a></li>
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<p style="text-indent: 30px; text-align:justify; font-family:Verdana, Times new roman, police3, police4;">Polyhydroxyalcanoates (PHAs) are universal prokaryotic storage compounds of carbon and
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energy. They are accumulated into the cell as spherical inclusion bodies in case of oversupply
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of carbon sources. We aim to control the <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage1/Theory">production</a> and the composition of granules in <span style="font-style:italic">Escherichia Coli</span>.
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Many industrial and biomedical applications for those granules had been evocated. However, we want to
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focus especially on two potential applications (see <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage2/Theory">Uses</a>):<br />
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<li>the granule as self-cleaving micro-beads in order to purify a recombinant protein of interest, see <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage2/Theory#anne3">Proteins purification</a></li>
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<li>the granule as a storage system for overproduced lipids of interest, for example DHA or EPA which have medical applications, see <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage2/Theory#anne2"> Storage of lipids of interest</a>.</li>
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We aim to regulate the granule production in <span style="font-style:italic">E. coli</span> thanks to two different <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage3/Strategy">strategies</a>: thermoregulation and chemoregulation (arabinose). However, our favorite new part is a strong promoter sensitive to temperature of the water bath (and also to shaking speed and osmolarity). </p><br><br>
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In bacteria, three separate monofunctional enzymes are required for PHA synthesis. In order to improve this pathway, we intend to <a href="https://2010.igem.org/Team:INSA-Lyon/Project/Stage1/Evolution">design a single multifunctional enzyme</a> based on the study of natural evolution of fatty acid synthesis in animals.</p><br>
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<p> Indeed, in plants and bacteria, the reactions involved in fatty acid synthesis also are catalyzed by separate monofunctional enzymes. But in animals, evolution has done synthetic biology and the different enzymes are integrated into a single multifunctional polypeptide in which substrates are handed from one functional domain to the next. We have begun to analyse the work of evolution in animals in order to design multifunctional bacterial enzymes that integrate discrete monofunctional enzymes. We hope that these enzymes could promote the synthesis of particular lipids, such as tri-acyl glycerols, and store these molecules into granules.</p>
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<p style="text-align:center;"><a href="#top">Top of Page</a></p>
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<p><strong>Did you know ?</strong></p>
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<h3>The <em>Droppy project</em> !</h3>
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<p> The great majority of microbes, simply cultivated in their usual media, are able to synthesize considerable amounts of intracellular lipids and store these molecules into large droplets easily stainable and observable. These spherical inclusions are surrounded by a mono-layer of lipids with embedded or attached proteins. To our knowledge, these lipid droplets (or "granules") had never been observed in wild-type <span style="font-style:italic">E. coli</span> strains. But recombinant strains have been constructed by cloning the genes responsible for the production of poly-hydroxybutyrate (PHB), an insoluble polyester, and these molecules were deposited into granules (with the diameter usually ranging between 100 and 500 nm and 5-10 granules per cell). These recombinant bacteria are able to accumulate as much as 80% of their dry weight in PHB.</p>
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<p> Considering this very important result, the working hypothesis at the origin of our project is that <span style="font-style:italic">E. coli</span> (and maybe any cell) is able to produce granules when large amounts of hydrophobic molecules are synthesized.</p>
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<p> In a first step, we will test this hypothesis by constructing <span style="font-style:italic">E. coli</span> strains which produce other hydrophobic molecules and examining granule synthesis. For this, we intend to construct a new part : a strong promoter sensitive to the shaking speed of the water bath in which the bacteria are cultured. Our objective is to trigger lipid synthesis and granule production just by varying this shaking speed.</p>
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<p> The second step of our project is the use of the PHB granules as a tool to produce and purify recombinant proteins. Various proteins, such as phasins, are attached to the external surface of the granules. Our objective is to construct a part able to target proteins of interest to this surface. Moreover, in order to facilitate purification, the sequence of a self-cleaving protein (intein) will be added to this part.</p>
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<p> In addition, we intend to develop a modelisation project. In plants and bacteria, the reactions involved in fatty acid synthesis are catalyzed by separate monofunctional enzymes. But in animals, evolution has done synthetic biology and the different enzymes are integrated into a single multifunctional polypeptide in which substrates are handed from one functional domain to the next. We have begun to analyse the work of evolution in animals in order to design multifunctional bacterial enzymes that integrate discrete monofunctional enzymes. We hope that these enzymes could promote the synthesis of particular lipids, such as tri-acyl glycerols, and store these molecules into granules.</p>
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Latest revision as of 21:35, 27 October 2010





The Droppy project !


Polyhydroxyalcanoates (PHAs) are universal prokaryotic storage compounds of carbon and energy. They are accumulated into the cell as spherical inclusion bodies in case of oversupply of carbon sources. We aim to control the production and the composition of granules in Escherichia Coli. Many industrial and biomedical applications for those granules had been evocated. However, we want to focus especially on two potential applications (see Uses):

  1. the granule as self-cleaving micro-beads in order to purify a recombinant protein of interest, see Proteins purification
  2. the granule as a storage system for overproduced lipids of interest, for example DHA or EPA which have medical applications, see Storage of lipids of interest.

We aim to regulate the granule production in E. coli thanks to two different strategies: thermoregulation and chemoregulation (arabinose). However, our favorite new part is a strong promoter sensitive to temperature of the water bath (and also to shaking speed and osmolarity).





In bacteria, three separate monofunctional enzymes are required for PHA synthesis. In order to improve this pathway, we intend to design a single multifunctional enzyme based on the study of natural evolution of fatty acid synthesis in animals.


Indeed, in plants and bacteria, the reactions involved in fatty acid synthesis also are catalyzed by separate monofunctional enzymes. But in animals, evolution has done synthetic biology and the different enzymes are integrated into a single multifunctional polypeptide in which substrates are handed from one functional domain to the next. We have begun to analyse the work of evolution in animals in order to design multifunctional bacterial enzymes that integrate discrete monofunctional enzymes. We hope that these enzymes could promote the synthesis of particular lipids, such as tri-acyl glycerols, and store these molecules into granules.


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