Difference between revisions of "Team:UChile-OpenBio"

 
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{{UChile-OpenBio}}
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            <img id="logo" alt="OpenBio Uchile PLA Project" src="https://static.igem.org/mediawiki/2015/b/b0/Cabecera.png" />
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                <ul>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio">HOME</a></li>
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                    <li>
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                        <a href="https://2015.igem.org/Team:UChile-OpenBio/Description">PROJECT</a>
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                        <ul>
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                            <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Description">PROJECT DESCRIPTION</a></li>
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                            <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Experiments">EXPERIMENT AND PROTOCOLS</a></li>
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                            <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Results">RESULTS</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Parts">PARTS</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Notebook">NOTEBOOK</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Collaborations">COLLABORATION</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Practices">HUMAN PRACTICES</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Safety">SAFETY</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Modelling">MODELLING</a></li>
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                    <li><a href="https://2015.igem.org/Team:UChile-OpenBio/Team">TEAM</a></li>
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            {{UChile-OpenBio}}
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<h2>Project description</h2>
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                <span class="titulo_seccion">Project</span>
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                <img alt="Project overview" src="https://static.igem.org/mediawiki/2015/d/d7/Uchile-Openbio_Home.png" usemap="#overview" />
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<h3>Overview of 2015 UChile-OpenBio Team Project</h3>
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Nowadays, fossil plastic contamination is still an issue. Each year, 130 million tons of plastics are produced in the world, which last between 500 to 1000 years in degrading, polluting our entire environment. A sustainable initiative is to produce biodegradable plastics, however the current synthesis process (chemical and biological) is complex and expensive. For this reason, the team UChile-OpenBio plans to engineer a biologic system, enabling it to degrade glucose in order to produce and export into the medium a biodegradable plastic called PLA. In the future the team hopes to replace the glucose for a renewable resource, the dun algae, which is located on the Chilean coasts. The aim is, in the long term, to implement a simple and cheaper system to produce bioplastic. This way, the team would help fighting the contamination due to fossil plastics.
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<h3>Background</h3>
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Each year, 130 million tons of plastics[1] are produced, which last between 500 to 1000 years in degrading[2]  and are responsible for the death of 1,5 millions of marine animals all around the world.[3] This corresponds to a Chilean waste of up to 25 thousand tons thrown into the ocean, where it can be brought back to the coast, sunk or accumulated near the Pascua Island.[4]  In Chile, the government emitted a ley project to forbid any sell of supermarket bags made of polyethylene, polypropylene and other artificial polymers non-biodegradable, which was accepted in the Patagonian territory last year. [5]
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<p class="imagen"><img src="https://static.igem.org/mediawiki/2015/4/43/Botellas.jpg" /><br><span class="caption">Representation of plastic contamination in the sea (ecologiaverde.com).</span></p>
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Fossil plastic contamination is not a new issue and several ways to reduce it have been explored. However, actions like recycling are not viable solutions, knowing that only up to the 30% of the produced plastics is actually reused. [6] A more sustainable initiative is to produce biodegradable plastics, made of renewable resources such as corn: their degradation time can be of only two years in the case of the PolyLactic Acid (PLA) and their physical properties are very similar to the classic plastic ones. [7] Nevertheless, the current synthesis, essentially driven by chemical reactions, is quite expensive because the process requires complex experimental conditions, for instance the absence of any trace of water, which raises the production costs.[2]
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This challenge, consisting in making the biodegradable plastic production cheaper, gave birth to another way to synthesise them: using microorganisms. Indeed, as it has been observed for the insulin, microorganisms have a great production capacity.[8] Several scientific studies already began to produce a bioplastic, PHB, with genetically modified bacterias.[9] The main difficulty resides in finding a way to export the bioplastic outside the cell. Indeed, the recuperation of cell products is a difficult and expensive challenge.
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Considering all these elements, the team UChile-OpenBio wants to reach, in the long term, the implementation of a secretory biological production of PLA from a renewable resource, the dun algae, which is located on the Chilean coasts. To do that, the first step consists in designing and testing the biologic system using a simple sugar: the glucose. This way, the team would help fighting the contamination due to fossil plastics.
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<h3>Main Goal</h3>
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For the iGEM competition, the team aims to engineer a biologic system, enabling it to degrade glucose in order to produce and export into the medium a biodegradable plastic called PLA.
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<h3>The project</h3>
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The team will start with the proof of concept, consisting in programming two populations of <em>Escherichia coli</em> to produce PLA from glucose. In a few words, the first population (in light green) will convert the glucose into an intermediate called lactate, which will be processed by the second population (in blue) to polymerize it into PLA.
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<p class="imagen"><img src="https://static.igem.org/mediawiki/2015/5/52/Schematic_process.jpg" /><br><span class="caption">A scheme representing the general process of our system.</span></p>
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The team is also implementing a pH-sensing system which will allow the bacteria to control the lactate production: the higher the intermediate concentration, the lower the pH, which induces a negative control in the first population of <em>E.coli</em>, stopping the production of lactate and by the way, of PLA. The second population of bacterias will possess an exportation system that would enable it to send the biologic PLA outside the cells, into the medium. This way, the purification of the bioplastic is easier.
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To ensure the safety of the persons working in the laboratory and of the environment, the team designed a safety system which consists in making arabinose-dependent the cell survival: while the bacterias grow up in laboratory conditions, defined by the presence of arabinose into the medium, the safety system is shut down. If the bacterias escape from their medium, the safety system won’t be turned off anymore and the cells will produce a toxin which will kill them.
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<h3>Potential Impact</h3>
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<h4>Advantages</h4>
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<ul>
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<li>Fossil plastics could be replaced in the long term by biodegradable plastics such as PLA, ending up with the environmental contamination associated.</li>
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<li>The cares provided by the medical area could be improved by the expansion of biocompatible prosthesis made of PLA.</li>
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<li>Digital fabrication laboratories could have better access to their feedstock (the PLA) which could promote the fabrication of Open Tools and the society empowering.</li>
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</ul>
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<h4>Desadvantages</h4>
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<ul>
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The team does not currently know what will be the nature of the produced PLA nor how will react the bacterias to the genetic modifications that have been made, so at least two consequences should be considered:
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<li>The biologic production of the PLA can lead to a non-utilizable bioplastic (physical state, purity, properties,…)</li>
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<li>Every engineered biologic system presents human and environmental risks and must be controlled by safety rules. However it is difficult to have a complete control over the biologic system, even with the safety system implemented (any aleatory mutation can inhibit it).</li>
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</ul>
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<h3>Future Prospects</h3>
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As said before, the project presented for the iGEM competition is only a proof concept: the team aims to bring the project beyond the academic field by changing the feedstock to a more sustainable one, the dun algae. What the team will do with the final project is still being discussed.
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<h3>Sponsors</h3>
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The team can already rely on the support of various institutions:
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<ul>
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<li>Centre for Biotechnology and Bioengineering</li>
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<li>Department of Chemical Engineering and Biotechnology</li>
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<li>National Committee of Scientific and Technologic Research</li>
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<li>Sigma-Aldrich Company</li>
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</ul>
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<h3>References</h3>
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<ol>
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<li>El Banco Mundial, 2014. Una bolsa de plástico para asfixiar el mar. [online] &lt;http://www.bancomundial.org/es/news/feature/2014/12/08/bolsa-de-plastico-asfixiar-planeta&gt; [consulted: 14-07-2015]</li>
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<li>Garlotta, 2002. A Literature Review of PolyLactic Acid. Journal of Polymers and the Environment, Vol. 9, No. 2.</li>
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<li>El Tiempo, 2014. Plásticos matan al año 1,5 millones de animales marinos. [online] &lt;http://www.eltiempo.com/estilo-de-vida/ciencia/muerte-de-animales-por-plasticos-lanzados-al-mar/14710998&gt; [consulted: 14-07-2015] </li>
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<li>La Tercera, 2015. Cristina Espinoza. Hasta 25 mil toneladas de plástico anuales se arrojan al mar desde Chile. [online] &lt;http://www.latercera.com/noticia/tendencias/2015/05/659-627978-9-hasta-25-mil-toneladas-de-plasticos-anuales-se-arrojan-al-mar-desde-chile.shtml&gt; [consulted: 14-07-2015]</li>
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<li>Chilean Senate, 2014. [online] &lt;http://www.senado.cl/prohibicion-de-bolsas-plasticas-en-la-patagonia-votaran-idea-de-legislar/prontus_senado/2014-10-23/122842.html&gt; [consulted: 14-07-2015] </li>
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<li>PlasticsEurope. Plásticos - Situación en 2011. Análisis de la producción, la demanda y la recuperación de plásticos en Europa en 2010. [online] &lt;http://www.plasticseurope.org/documents/document/20111107102611-pe_factsfigures_es_2011_lr_final041111.pdf&gt; [consulted: 15-07-2015]</li>
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                <map name="overview">
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                    <area shape="circle" coords="183,290,60" alt="Overview" href="https://2015.igem.org/Team:UChile-OpenBio/Description"/>
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                    <area shape="circle" coords="620,150,60" alt="Overview" href="https://2015.igem.org/Team:UChile-OpenBio/Notebook"/>
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                    <area shape="circle" coords="700,450,60" alt="Overview" href="https://2015.igem.org/Team:UChile-OpenBio/Attributions"/>
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                    <area shape="circle" coords="280,61,60" alt="Specifics goals" href="https://2015.igem.org/Team:UChile-OpenBio/Collaborations" />
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                    <area shape="circle" coords="100,350,60" alt="Specifics goals" href="https://2015.igem.org/Team:UChile-OpenBio/Practices" />
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                    <area shape="circle" coords="725,67,60" alt="Main goal" href="https://2015.igem.org/Team:UChile-OpenBio/Safety" />
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                    <area shape="circle" coords="485,67,60" alt="Background" href="https://2015.igem.org/Team:UChile-OpenBio/Modeling" />
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                    <area shape="circle" coords="485,407,60" alt="Experiment" href="https://2015.igem.org/Team:UChile-OpenBio/Team" />
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                </map>
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<img id="bacteria_con_capa" class="bottom right" alt="Super bacterias" src="https://static.igem.org/mediawiki/2015/9/94/Uchile-Openbio_hug.jpeg" /> <br></article><br>
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</section>
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<section id="background">
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<div class="division">
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<span class="titulo_seccion" >Overview: Background</span> 
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<article>                                       
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                        <p>Each year, 130 million tons of fossil plastics are produced in the world, which take 500-1000 years to degrade, and pollute the environment; 1,5 millions of marine animals were killed in 2014 and we will eventually kill us too (Figure 1, Project). A sustainable initiative is to produce biodegradable plastics; however its synthesis process (chemical and biological) is complex and expensive. The team UChile-OpenBio designed two populations of bacteria to achieve this: the  team will use Escherichia coli to produce a biodegradable plastic called PLA (Polylactic acid) from easy to assimilate renewable resources. The first population will convert glucose into lactate and will self-regulate its production by sensing the pH. The second population will polymerize lactate into PLA and will export it into the medium. In addition the team is planning to replace the glucose by Chilean brown macroalgae (kelp), a renewable resource to sustainably produce PLA. In this way, the team would help fighting against pollution, contributing to a better world! </p>
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<li>Serna et al. Ácido Poliláctico (PLA): Propiedades y Aplicaciones. Ingeniería y Competitividad (2003), Vol.5, 16-26.</li>
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                    </div>
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                </article>
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                <article>
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                    <div class="division">
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                        <div class="half">
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                            <h1>General Concepts</h1>
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                            <p>A genetic module is constituted of 4 elements, called parts, that are DNA sequences which possess a specific function: the promotor, the RBS, the coding sequence and the terminator.</p>
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                            <p>The role of a genetic module is to produce certain quantity of a protein under some specific conditions. The fabrication process, from DNA to protein, consists in two steps: <span class="destacado">transcription</span> and <span class="destacado">translation</span>.</p>
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                            <p>If the DNA is a book of recipes, the transcription process consists in photocopying one page, in which are written the instructions for one specific food; and the translation process corresponds to the cooking phase, in which the "cooker" is the ribosome. The ribosome reads the instructions and at the same time builds the protein. This is called the <span class="destacado">Central Dogma of Biology</span>.</p>
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                        </div>
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                        <div class="half last">
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                            <img src="https://static.igem.org/mediawiki/2015/3/38/UChile_OpenBio_GeneralConcepts.png">
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                            <label> Scheme of the general concepts </label>
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                            <img src="https://static.igem.org/mediawiki/2015/d/d2/UChile_OpenBio_GeneralConcepts_Sev.png">
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                        </div>
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                    </div>
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                </article>
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                <article>
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                    <div class="division">
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                        <div class="half">
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                            <h1>General Concepts</h1>
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                            <p>The transcription can be regulated by different factors (repressors, pH, light, etc) which are detected by the <span class="destacado">promotor</span>. These signals induce or inhibit the activation of the module.</p>
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                            <p>If the module is activated then the transcription process begins, in which several copies of the message are made.</p>
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                            <p>The <span class="destacado">RBS</span> (Ribosomal Bonding Site) intervenes in the translation process: if it is strong, many proteins will be synthesised. If it is weak, few proteins will be synthetized. So the function of RBS is regulate the quantity of produced proteins.</p>
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                            <p>The <span class="destacado">coding sequence</span> is the message, the recipe that shall be translated to protein.</p>
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                            <p>The <span class="destacado">terminator</span> is a DNA sequence that puts an end to the transcription process.</p>
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                        </div>
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                        <div class="half last">
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                            <img class="" alt="Genetic module and parts in legos" src="https://static.igem.org/mediawiki/2015/f/f2/Description-genetic_module_parts_legos.png" />
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                            <img src="https://static.igem.org/mediawiki/2015/6/60/UChile_OpenBio_GeneralConcepts_Lucho.png">
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                        </div>
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                    </div>
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                </article>
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                <article>
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                    <h1>Why Legos are a friendly way to explain SB?</h1>
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                    <div class="division">
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                        <div class="half">
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                            <p>The team decided to used legos to explain the pla project due to different reasons.</p>
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                        </div>
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                    </div>
  
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<li>Jong et al. Production of recombinant proteins by high cell density culture of Escherichia coli. Chemical Engineering Science (2006). Vol. 61, Issue 3, 876–885. </li>
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                    <div class="division">
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                        <div class="one_third">
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                            <h2>First Reason</h2>
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                            <p>They make tangible microscopic things</p>
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                            <img src="https://static.igem.org/mediawiki/2015/7/7f/UChile_OpenBio_FirstReason.png" >
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                        </div>
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                        <div class="one_third">
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                            <h2>Second Reason</h2>
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                            <p>They allow a strong analogy with the DNA parts called « bio-bricks»</p>
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                            <p>They are standard and modular like a biobricks</p>
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                            <img src="https://static.igem.org/mediawiki/2015/9/9a/UChile_OpenBio_SecondReason.png">
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                            <p> <a href="https://www.flickr.com/photos/mknowles/47457221"> Source </a>
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                        </div>
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                        <div class="one_third last">
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                            <h2>Third Reason</h2>
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                            <p>Finally, they are a funny way to explain complex concepts... for the students...and the teachers!</p>
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                            <img src="https://static.igem.org/mediawiki/2015/e/ee/UChile_OpenBio_3rdReason.png" align=left>
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                            <img src="https://static.igem.org/mediawiki/2015/9/90/UChile_OpenBio_3rdReason_2.png" align=right >
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                        </div>
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                    </div>
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                </article>
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            </section>
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            <section id="lego_description">
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                <span class="titulo_seccion">Overview: Lego description</span>
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<li>Mahishi et al. Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: Effect of various carbon and nitrogen sources. Microbiol. Res. (2003) 158, 19–27</li>
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<img class="" alt="Simbologiía" src="https://static.igem.org/mediawiki/2015/f/f7/Uchile-Openbio_for_legos.jpeg" />
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                <article>
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                    <div class="division">
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                            <h3>E. Coli 1 Population: “Lactadora”</h3>
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                            <img class="" alt="Lactadora" src="https://static.igem.org/mediawiki/2015/0/0b/Lego_description-bacteria_lego.png" />
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                            <p>In our project we used two bacteria « Escherichia coli »: name and first name of the bacteria.<br>This population are responsible for lactate production.</p>
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                        </div>
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                        <div class="half last">
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                            <h3>E. Coli 2 Population: “PLAdora”</h3>
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                            <img class="" alt="PLAdora" src="https://static.igem.org/mediawiki/2015/0/0b/Lego_description-bacteria_lego.png" />
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                            <p>« Escherichia coli »: name and first name of the bacteria. « 1 » y « 2 » only mean that these bacteria, in this process, are responsible for different functions (but they belong the same bacteria strain).<br>This population are responsible for process the lactate into PLA.</p>
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                        </div>
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                    </div>
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                </article>
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                        <div class="two_third">
 +
                            <img class="" alt="LDH" src="https://static.igem.org/mediawiki/2015/7/7f/Lego_description-LDH.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            The « Lactate DeHydrogenase » (LDH) is responsible for lactate production.
 +
                        </div>
 +
                    </div>
 +
                    <img class="" alt="Scheme" src="https://static.igem.org/mediawiki/2015/6/64/Lego_description-esquema.png" />
 +
                    <div id="esquema_descripcion" class="division">
 +
                        <div class="one_third">
 +
                            <p>Transformation of glucose into pyruvate is a bacterial natural process, it doesn’t need human intervention.</p>
 +
                        </div>
 +
                        <div class="one_third">
 +
                            <p>LDH uses pyruvate and transforms it into lactate, the intermediate compound that allows PLA synthesis.</p>
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            <p>Finally, lactate can be obtained from glucose thanks to LDH, which gene we insert into the bacterium.</p>
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                            <img class="" alt="Lactate" src="https://static.igem.org/mediawiki/2015/9/90/Lego_description-lactate.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            According to last image, the module which produces LDH is indirectly responsible for lactate production, for it will be symbolized as if the LDH directly produced lactate.
 +
                        </div>
 +
                    </div>
 +
                    <img class="" alt="Lactate" src="https://static.igem.org/mediawiki/2015/b/b6/Lego_description-lactate_populations.png" />
 +
                    <div class="division">
 +
                        <div class="half">
 +
                            <p>The lactate produced by the <span class="destacado">E.coli 1 Population</span> is secreted into the medium and enters in the bacteria of the E.coli 2 Population</p>
 +
                        </div>
 +
                        <div class="half last">
 +
                            <p>Lactate enters into the <span class="destacado">E.coli 2 Population</span> to be processed into PLA by the presented module.</p>
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                            <img class="" alt="PhaC" src="https://static.igem.org/mediawiki/2015/3/3f/Lego_description-phac.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            The lactate convertion into PLA is driven by two enzymes: « P-CoA-T »: Propionyl-CoA-Transferase « phaC1 »: PolyHydroxy Alkanoate –Class 1
 +
                        </div>
 +
                    </div>
 +
                    <img class="" alt="PLA" src="https://static.igem.org/mediawiki/2015/0/0d/Lego_description-lactato_pla.png" />
 +
                    <div id="esquema_descripcion" class="division">
 +
                        <div class="one_third">
 +
                            <p>The P-CoA-T unites lactate with a cofactor called « Coenzyme A », transforming lactate into « Lactyl-CoA ».</p>
 +
                        </div>
 +
                        <div class="one_third">
 +
                            <p>PhaC1 gathers all the lactyl-CoA that are present in the cell and unites them together. . .</p>
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            <p>This reaction is called « polymerization ». The product of this polymerization is the PLA.</p>
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <img class="" alt="Lactate to PLA" src="https://static.igem.org/mediawiki/2015/3/38/Lego_description-sumpup_lactate_to_pla.png" />
 +
                    <div class="division">
 +
                        <div class="half">
 +
                            <p>To sum up, this module processes lactate to transform it into PLA.</p>
 +
                        </div>
 +
                        <div class="half last">
 +
                            <p>The produced PLA is processed by a second module, which produces a hybrid protein constituted of a first protein called phasyn and of a much smaller protein having affinity for PLA. The role of this hybrid protein is to make the cell know that PLA is ready for exportation.</p>
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <img class="" alt="PLA ready to export" src="https://static.igem.org/mediawiki/2015/f/f3/Lego_description-pla_export.png" />
 +
                    <p class="harto_margen">The hybrid protein sticks with PLA and the cell exports it into the extra-cellular medium, ready to be extracted and purified.</p>
 +
                </article>
 +
                <article>
 +
                    <h3>Important Modules of the process</h3>
 +
                    <div class="division">
 +
                        <div class="half">
 +
                            <h3>E.coli 1 Population</h3>
 +
                        </div>
 +
                        <div class="half last">
 +
                            <h3>E.coli 2 Population</h3>
 +
                        </div>
 +
                    </div>
 +
                    <img class="" alt="E1 E2 population" src="https://static.igem.org/mediawiki/2015/4/49/Lego_description-important_modules.png" />
 +
                    <div class="division">
 +
                        <div class="half">
 +
                            <div class="division">
 +
                                <div class="half centered">
 +
                                    Glucose transformation into lactate
 +
                                </div>
 +
                            </div>
 +
                        </div>
 +
                        <div class="half last">
 +
                            <div class="division">
 +
                                <div class="one_third centered">
 +
                                    Transformation of lactate into PLA
 +
                                </div>
 +
                                <div class="one_third centered">
 +
                                    PLA exportation
 +
                                </div>
 +
                            </div>   
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <h3>Regulation of lactate production in E.coli 1</h3>
 +
                   
 +
               
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                        <img class="" alt="Regulation E1" src="https://static.igem.org/mediawiki/2015/c/cb/Lego_description-regulation_E1.png" /> 
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            When being outside the cells, lactate acidifies the medium, which is dangerous for the bacteria. That’s why a pH-sensing module has been built to detect pH levels and produce the TetR protein when the pH is lower than 5.5.
 +
                        </div>
 +
                    </div>
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                            <img class="" alt="Regulation E1" src="https://static.igem.org/mediawiki/2015/3/39/Lego_description-ph_increase.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            TetR is a repressor of the lactate production module. It means, when pH becomes lower than 5.5, the lactate production is shut off by TetR. This shrinks the lactate concentration in the medium so that the pH increases again.
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <h3>Communication between E.coli 1 and E.coli 2</h3>
 +
                    <img class="" alt="Regulation E1" src="https://static.igem.org/mediawiki/2015/a/af/Lego_description-communication.png" />
 +
                    <p>In fact, the module that produces lactate not only produces it but is also responsible for the synthesis of a small molecule called HSL (HomoSeryl-Lactone) . This molecule is exported into the medium and is able to enter the second bacterium, being responsible for communication between E.coli 1 and 2.</p>
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                            <img class="" alt="Regulation E2" src="https://static.igem.org/mediawiki/2015/3/31/Lego_description-hsl.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            Inside the E.coli 2 bacteria, is also inserted a module which produces a protein called LuxR.
 +
                        </div>
 +
                    </div>
 +
                    <div class="division">
 +
                        <div class="two_third">
 +
                            <img class="" alt="Regulation E2" src="https://static.igem.org/mediawiki/2015/3/33/Lego_description-luxr.png" />
 +
                        </div>
 +
                        <div class="one_third last">
 +
                            The concerted action of luxR with HSL activates the other modules of the E.coli 2 (logic door « AND »),
 +
                        </div>
 +
                    </div>
 +
                </article>
 +
                <article>
 +
                    <h3>Communication between E.coli 1 and E.coli 2</h3>
 +
                    <img class="" alt="Communication between" src="https://static.igem.org/mediawiki/2015/c/c8/Lego_description-between.png" />
 +
                    <p>The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.</p>
 +
                    <img class="" alt="Communication global" src="https://static.igem.org/mediawiki/2015/a/a0/Lego_description-global.png" />
 +
                    <p>The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.</p>
 +
                </article>
 +
            </section>
 +
 
 +
    <footer>
 +
        <h1>Our Sponsors</h1>
 +
        <img src="https://static.igem.org/mediawiki/2015/4/4f/Footer_sponsors.png" alt="UChile Sponsors" />
 +
    </footer>
 +
        </div> <!-- #contentContainer -->
 +
    </body>
 
</html>
 
</html>

Latest revision as of 15:54, 14 November 2015


TODO supply a title

Project Project overview Overview Overview Overview Specifics goals Specifics goals Main goal Background Experiment Super bacterias

Overview: Background

Each year, 130 million tons of fossil plastics are produced in the world, which take 500-1000 years to degrade, and pollute the environment; 1,5 millions of marine animals were killed in 2014 and we will eventually kill us too (Figure 1, Project). A sustainable initiative is to produce biodegradable plastics; however its synthesis process (chemical and biological) is complex and expensive. The team UChile-OpenBio designed two populations of bacteria to achieve this: the team will use Escherichia coli to produce a biodegradable plastic called PLA (Polylactic acid) from easy to assimilate renewable resources. The first population will convert glucose into lactate and will self-regulate its production by sensing the pH. The second population will polymerize lactate into PLA and will export it into the medium. In addition the team is planning to replace the glucose by Chilean brown macroalgae (kelp), a renewable resource to sustainably produce PLA. In this way, the team would help fighting against pollution, contributing to a better world!

General Concepts

A genetic module is constituted of 4 elements, called parts, that are DNA sequences which possess a specific function: the promotor, the RBS, the coding sequence and the terminator.

The role of a genetic module is to produce certain quantity of a protein under some specific conditions. The fabrication process, from DNA to protein, consists in two steps: transcription and translation.

If the DNA is a book of recipes, the transcription process consists in photocopying one page, in which are written the instructions for one specific food; and the translation process corresponds to the cooking phase, in which the "cooker" is the ribosome. The ribosome reads the instructions and at the same time builds the protein. This is called the Central Dogma of Biology.

General Concepts

The transcription can be regulated by different factors (repressors, pH, light, etc) which are detected by the promotor. These signals induce or inhibit the activation of the module.

If the module is activated then the transcription process begins, in which several copies of the message are made.

The RBS (Ribosomal Bonding Site) intervenes in the translation process: if it is strong, many proteins will be synthesised. If it is weak, few proteins will be synthetized. So the function of RBS is regulate the quantity of produced proteins.

The coding sequence is the message, the recipe that shall be translated to protein.

The terminator is a DNA sequence that puts an end to the transcription process.

Genetic module and parts in legos

Why Legos are a friendly way to explain SB?

The team decided to used legos to explain the pla project due to different reasons.

First Reason

They make tangible microscopic things

Second Reason

They allow a strong analogy with the DNA parts called « bio-bricks»

They are standard and modular like a biobricks

Source

Third Reason

Finally, they are a funny way to explain complex concepts... for the students...and the teachers!

Overview: Lego description Simbologiía

E. Coli 1 Population: “Lactadora”

Lactadora

In our project we used two bacteria « Escherichia coli »: name and first name of the bacteria.
This population are responsible for lactate production.

E. Coli 2 Population: “PLAdora”

PLAdora

« Escherichia coli »: name and first name of the bacteria. « 1 » y « 2 » only mean that these bacteria, in this process, are responsible for different functions (but they belong the same bacteria strain).
This population are responsible for process the lactate into PLA.

LDH
The « Lactate DeHydrogenase » (LDH) is responsible for lactate production.
Scheme

Transformation of glucose into pyruvate is a bacterial natural process, it doesn’t need human intervention.

LDH uses pyruvate and transforms it into lactate, the intermediate compound that allows PLA synthesis.

Finally, lactate can be obtained from glucose thanks to LDH, which gene we insert into the bacterium.

Lactate
According to last image, the module which produces LDH is indirectly responsible for lactate production, for it will be symbolized as if the LDH directly produced lactate.
Lactate

The lactate produced by the E.coli 1 Population is secreted into the medium and enters in the bacteria of the E.coli 2 Population

Lactate enters into the E.coli 2 Population to be processed into PLA by the presented module.

PhaC
The lactate convertion into PLA is driven by two enzymes: « P-CoA-T »: Propionyl-CoA-Transferase « phaC1 »: PolyHydroxy Alkanoate –Class 1
PLA

The P-CoA-T unites lactate with a cofactor called « Coenzyme A », transforming lactate into « Lactyl-CoA ».

PhaC1 gathers all the lactyl-CoA that are present in the cell and unites them together. . .

This reaction is called « polymerization ». The product of this polymerization is the PLA.

Lactate to PLA

To sum up, this module processes lactate to transform it into PLA.

The produced PLA is processed by a second module, which produces a hybrid protein constituted of a first protein called phasyn and of a much smaller protein having affinity for PLA. The role of this hybrid protein is to make the cell know that PLA is ready for exportation.

PLA ready to export

The hybrid protein sticks with PLA and the cell exports it into the extra-cellular medium, ready to be extracted and purified.

Important Modules of the process

E.coli 1 Population

E.coli 2 Population

E1 E2 population
Glucose transformation into lactate
Transformation of lactate into PLA
PLA exportation

Regulation of lactate production in E.coli 1

Regulation E1
When being outside the cells, lactate acidifies the medium, which is dangerous for the bacteria. That’s why a pH-sensing module has been built to detect pH levels and produce the TetR protein when the pH is lower than 5.5.
Regulation E1
TetR is a repressor of the lactate production module. It means, when pH becomes lower than 5.5, the lactate production is shut off by TetR. This shrinks the lactate concentration in the medium so that the pH increases again.

Communication between E.coli 1 and E.coli 2

Regulation E1

In fact, the module that produces lactate not only produces it but is also responsible for the synthesis of a small molecule called HSL (HomoSeryl-Lactone) . This molecule is exported into the medium and is able to enter the second bacterium, being responsible for communication between E.coli 1 and 2.

Regulation E2
Inside the E.coli 2 bacteria, is also inserted a module which produces a protein called LuxR.
Regulation E2
The concerted action of luxR with HSL activates the other modules of the E.coli 2 (logic door « AND »),

Communication between E.coli 1 and E.coli 2

Communication between

The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.

Communication global

The concerted action of LuxR with HSL activates the PLA production and makes possible its exportation. This activation is possible only if there is enough lactate in the medium (since it is the same module that produces lactate and HSL). This way, E.coli 1 leads the production, telling to E.coli 2 when the PLA synthesis can begin). That’s why it is said that HSL is a communication molecule.

Our Sponsors

UChile Sponsors