Difference between revisions of "Team:UChile OpenBio-CeBiB"

 
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<h1> Greenhardtii Project </h1>
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<p> Have you ever looked up at the sky and feel upset about its grayish color? Even in summer's brightest days you can see a lot of smog all around the city, not to mention the shocking view from our highest hills that give the sensation of being embraced by an everlasting cloud of uncertainty about our future. When will it end? We don´t know. How can we avoid this? To that we all have an answer, but constant industrial recklessness makes it pretty difficult to actually change anything. What can we do to help? Well, that's what Greenhardtii Project is about: we want to reduce carbon dioxide emissions by the means of the ability of microalgae to capture this problematic gas and use it to produce materials of interest. To make it even better,
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we will artificially increase its carbon uptake capacity and hinder the natural synthesis of starch to concentrate glucose availability. </p>
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<h2> Overview </h2>
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<p>The microorganism that we use is <i>Chlamydomonas reinhardtii</i>, a microalgae extensively used for genetic studies and algae metabolism.</p>
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<p>In regard to the increase in carbon uptake capacity, we focus in the Calvin cycle and consider the possibility of an additional enzyme to accelerate the process. Specifically, we evaluated the expected effects of incorporating FBP/SBPase to this metabolic route. Moreover, we ran short simulations on the kinetics of each reaction involved in the cycle and the consequences of modifying the parameters of the enzyme involved in them. We then came to the conclusion that adding FBP/SBPase is feasible. To allow a constant genetic expression of the FBP/SBPase gene we decided to use a strongly constitutive (but enhanceable with light and temperature) fusion promoter HSP70A/RBCS2, along with the NOST terminator and a chloroplast peptide signal (cTP).</p>
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<p>To hinder the synthesis route of starch, we plan to control the deactivation of genes STA1 and STA2 to prevent expression of Glucose-1-phosphate adenylyltransferase enzyme that catalyzes the conversion of Glucose-1-phosphate to ADP-glucose, a compound prior to amylose (which is the precursor of starch), and the deactivation of gene GBS2 that encodes the enzyme Granule Bound Starch Synthase, avoiding the transformation of Glucose-1-phosphate to UDP-glucose. To do such regulation we want to incorporate antisense DNA, that way the RNA transcribed will be complimentary to the RNA of the original genes, causing them to bind and block (partially or completely) the translation process. The promoters used for these will be the B12-responsive MetE. Even though it is known that the use of vitamin B12 for a constant regulation of a microalgae culture is not economically feasible, its use will be for evaluations of the genetic circuit only. In a further development of the project we will change to a different promoter, possibly a light-induceable one. The terminator will also be NOST.</p>
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<p>Here we present an image that summarizes our genetic circuit:</p>
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<p>[[insertar imagen del circuito]]</p>
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<h2> Lab Procedures and Experimental Design </h2>
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<p>To verify correct expression of the FBP/SBPase protein, a fusion with the red fluorescent protein RFP is planned. So, the construct of the gene to insert goes as follows:</p>
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<h5> HSP70A/RBCS2-cTP-FBP/SBPase-RFP-NOST </h5>
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<p>Afterwards, ligation to a plasmid must be made. We are evaluating the possibility of using pCAMBIA1301 or pCAMBIA1304.</p>
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<p>Once the insertion has been made, the plasmids must be analyzed in electrophoresis. For this, we must run a sample of ligated plasmid, unligated lasmid, ladder and solution without plasmid. A PCR is also a viable option.</p>
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<p>Then, extraction of the resulting bands from the electrophoretic gel shall be done.</p>
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<p>Once the plasmids with correct insertion of the gene has been successfully verified, transformation of <i>E. coli</i> will be done to amplify the availibility of the modified vector. To make this possible, LB culture medium must be prepared. Afterwards, electroporation of the bacteria will be conducted to ensure plasmid flux through <i>E. coli</i>, though chemical methods of transformation are also being discussed. Then, the bacteria will be transferred to selective medium in order to distinguish transformed and non-transformed colonies, probably containing kanamycin and Xgal. Once a sufficient amount of <i>E. coli</i> population is achieved, DNA material is then extracted and purified to obtain the desired vector.</p>
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<h5> Transformation of <i>Chlamydomonas reinhardtii</i></h5>
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<p>Two types of transformation methods are currently being evaluated: the traditional and low-efficient glass bead method, and the use of <i>Agrobacterium tumefaciens</i>.</p>
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<h4> About the hindering mechanism of starch synthesis</h4>
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<p>As told previously, this objective will be achieved through the use of antisense DNA. The genes to be inserted would be:</p>
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<h5> MetE-antiSTA1-NOST, MetE-antiSTA6-NOST, MetE-antGBS2-NOST</h5>
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<p> Experimental procedure for the transformation process is analogous to the recently described one. However, the correct sequence of the gene is still being evaluated.</p>
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<h2> Mathematical Modelling </h2>
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<p> [[bla bla bla]] </p></div>
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<h2> Human Practices </h2>
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<h5> Strategy </h5>
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Investigate how much does Chilean people know about contamination, science, biotechnology, eco-friendly production, public spaces, etc. This info will be obtained through several surveys conducted in Santiago de Chile (capital). In response to the information obtained we will start to empower the people by doing workshops, focus groups, talks, visits to laboratories, etc. and design interesting proposal for them.
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As a general result of all the surveys, we want to model a couple of bioreactors with friendly shapes for people in order to be emplaced in public spaces such as squares, streets, parks, museums, highway, etc. </p>
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<h2> Financial Resources </h2>
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<p> [[bla bla bla]] </p></div>
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<h2> Team Background </h2>
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<p> [[descripción de nosotros]] </p></div>
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<h1> Welcome to iGEM 2017! </h1>
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<p>Your team has been approved and you are ready to start the iGEM season! </p>
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<h5>Before you start: </h5>
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<p> Please read the following pages:</p>
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<li>  <a href="https://2017.igem.org/Competition">Competition Hub</a> </li>
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<li> <a href="https://2017.igem.org/Competition/Deliverables/Wiki">Wiki Requirements page</a></li>
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<li> <a href="https://2017.igem.org/Resources/Template_Documentation">Template documentation</a></li>
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<h5> Styling your wiki </h5>
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<p>You may style this page as you like or you can simply leave the style as it is. You can easily keep the styling and edit the content of these default wiki pages with your project information and completely fulfill the requirement to document your project.</p>
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<p>While you may not win Best Wiki with this styling, your team is still eligible for all other awards. This default wiki meets the requirements, it improves navigability and ease of use for visitors, and you should not feel it is necessary to style beyond what has been provided.</p>
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<h5> Wiki template information </h5>
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<p>We have created these wiki template pages to help you get started and to help you think about how your team will be evaluated. You can find a list of all the pages tied to awards here at the <a href="https://2017.igem.org/Judging/Pages_for_Awards">Pages for awards</a> link. You must edit these pages to be evaluated for medals and awards, but ultimately the design, layout, style and all other elements of your team wiki is up to you!</p>
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<h5> Editing your wiki </h5>
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<p>On this page you can document your project, introduce your team members, document your progress and share your iGEM experience with the rest of the world! </p>
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<p> <a href="https://2017.igem.org/wiki/index.php?title=Team:Example&action=edit"> </a>Use WikiTools - Edit in the black menu bar to edit this page</p>
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<h5>Tips</h5>
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<p>This wiki will be your team’s first interaction with the rest of the world, so here are a few tips to help you get started: </p>
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<li>State your accomplishments! Tell people what you have achieved from the start. </li>
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<li>Be clear about what you are doing and how you plan to do this.</li>
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<li>You have a global audience! Consider the different backgrounds that your users come from.</li>
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<li>Make sure information is easy to find; nothing should be more than 3 clicks away.  </li>
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<li>Avoid using very small fonts and low contrast colors; information should be easy to read.  </li>
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<li>Start documenting your project as early as possible; don’t leave anything to the last minute before the Wiki Freeze. For a complete list of deadlines visit the <a href="https://2017.igem.org/Calendar">iGEM 2017 calendar</a> </li>
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<li>Have lots of fun! </li>
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<h5>Inspiration</h5>
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<p> You can also view other team wikis for inspiration! Here are some examples:</p>
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<li> <a href="https://2014.igem.org/Team:SDU-Denmark/"> 2014 SDU Denmark </a> </li>
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<li> <a href="https://2014.igem.org/Team:Aalto-Helsinki">2014 Aalto-Helsinki</a> </li>
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<li> <a href="https://2014.igem.org/Team:LMU-Munich">2014 LMU-Munich</a> </li>
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<li> <a href="https://2014.igem.org/Team:Michigan"> 2014 Michigan</a></li>
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<li> <a href="https://2014.igem.org/Team:ITESM-Guadalajara">2014 ITESM-Guadalajara </a></li>
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<li> <a href="https://2014.igem.org/Team:SCU-China"> 2014 SCU-China </a></li>
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<h5> Uploading pictures and files </h5>
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<p> You can upload your pictures and files to the iGEM 2017 server. Remember to keep all your pictures and files within your team's namespace or at least include your team's name in the file name. <br />
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When you upload, set the "Destination Filename" to <br><code>T--YourOfficialTeamName--NameOfFile.jpg</code>. (If you don't do this, someone else might upload a different file with the same "Destination Filename", and your file would be erased!)<br><br>
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UPLOAD FILES
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Latest revision as of 22:56, 14 December 2017

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