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| - | <h2>Project description</h2> | + | <h2>We had a dream about fully standardized parts</h2> |
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| | <!-- <div class="note">Here goes something about our project</div> --> | | <!-- <div class="note">Here goes something about our project</div> --> |
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| - | Our project is divided in following parts:
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| - | <li><a href="https://2010.igem.org/Team:Warsaw/Stage1/RBSMeas">RBS Measurement</a></li>
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| - | <li><a href="https://2010.igem.org/Team:Warsaw/Stage1/PromMeas">Promoter Measurement</a></li>
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| - | <li>MinC based <a href="https://2010.igem.org/Team:Warsaw/Stage2">kill switch</a></li>
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| - | <li><a href="https://2010.igem.org/Team:Warsaw/Stage3">BactoDHL</a> - universal platform of protein and DNA delivery to mammalian cells based on invasive coli strain</li>
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| - | <!--<li>ColiStainer - an implementation of BactoDHL which stains various compartments of alive mammalian cells using fluorescent proteins fused with localization signals</li>-->
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| - | <div class="note">RBS Measurement</div> | + | <div class="note">Introduction</div> |
| - | <div>In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2010 spring distribution. Our list of measured parts includes RBSs both from Community and Anderson's collections. <br>We have used standard measurement kit composed of promoter BBa_J23100 and GFP+terminator part BBa_I130401. All measurements were carried out using flow cytometer. <br>Results are <a href="https://2010.igem.org/Team:Warsaw/Stage1/RBSMeas">here</a></div> | + | <div align="justify"> |
| | + | So far all the RBS parts were measured with GFP. We assumed that the strength of RBS is standard – does not depend on the expressed protein. But it turns out that the world is not as perfect as we would like it to be: it was during the 2010 iGEM competition that we came across the first sings that something is wrong. To investigate this problem further, this year we measured several RBS parts with various fluorescent proteins. Unfortunately, we were right. |
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| | + | <div class="note">The Problem</div> |
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| | + | Variances in strength of RBS parts are caused by different mRNA fold. The beginning of every protein influences mRNA fold, which in turn makes the RBS sequence more or less accessible for the ribosome. If the RBS becomes a part of a secondary structure, ribosome is less likely to bind to it and the protein expression is limited or even impossible. |
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| - | <div class="note">Promoter Measurement</div>
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| - | <div>Additionally we have defined <a href="http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_58:_Absolute_measurement_of_bacterial_promoter_strength_in_cell-free_system_by_qPCR">new standard of absolute promoter strength measurement (RFC58)</a>. <br>Using it we have measured <a href="http://partsregistry.org/Part:BBa_J23100">J23100</a> and <a href="http://partsregistry.org/Part:BBa_I719005">pT7</a>. <br>Results are <a href="https://2010.igem.org/Team:Warsaw/Stage1/PromMeas">here</a></div>
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| - | <div class="note">ExpressIt vector family</div>
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| - | We have used measurement results to create <a href="https://2010.igem.org/Team:Warsaw/Stage1/ExpressIt">ExpressIt family of vectors with defined expression strength</a>.
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| - | <div class="note">MinC based kill sitch</div>
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| - | <div>To make our invasive E. coli strain safe we are planning to use division inhibitor protein MinC from E. coli BL21. Our <a href="https://2010.igem.org/Team:Warsaw/Stage2/Results">results</a> show that expression of MinC efficiently blocks E. coli cell division without impairing other functions such as protein synthesis. </div>
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| - | <div class="note">BactoDHL</div>
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| - | <div align="justify">BactoDHL is a universal platform of protein and DNA delivery to mammalian cells. It's based on E. coli strain that expresses invasion determinants: invasin from Yersinia pestis and listeriolysin (LLO) from Listeria monocytogenes. Invasin causes uptake of the bacterium into the mammalian cell by induction of clatrin-mediated endocytosis. Bacterial cells are lysed in the endosome and then LLO is relased. LLO is a pore-forming toxin which causes endosomal membrane disruption and release of the payload (either protein or DNA) into cytoplasm of the mammalian cell.<br>To see BactoDHL in action look <a href="https://2010.igem.org/Team:Warsaw/Stage3/Results">here</a></div>
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| - | <div class="note">ColiStainer</div>
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| - | <div><b>We have planned to build a ColiStainer strain. Unfortunately there was not enough time to accomplish this task. Below is original information about ColiStainer concept.</b><br> ColiStainer is a BactoDHL strain expressing various fluorescence proteins (i.e. GFP) fused to various cell localization signals. When ColiStainer invades mammalian cells it releases payload of those proteins, which are transported to various cellular compartments (i.e. mitochondria, nucleus or Golgi apparatus). It causes the compartments to glow in various colors and produces cheaper and easier equivalent of immunohistochemical stain with florophore-conjugated antibodies.</div>
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We had a dream about fully standardized parts
Introduction
So far all the RBS parts were measured with GFP. We assumed that the strength of RBS is standard – does not depend on the expressed protein. But it turns out that the world is not as perfect as we would like it to be: it was during the 2010 iGEM competition that we came across the first sings that something is wrong. To investigate this problem further, this year we measured several RBS parts with various fluorescent proteins. Unfortunately, we were right.
The Problem
Variances in strength of RBS parts are caused by different mRNA fold. The beginning of every protein influences mRNA fold, which in turn makes the RBS sequence more or less accessible for the ribosome. If the RBS becomes a part of a secondary structure, ribosome is less likely to bind to it and the protein expression is limited or even impossible.
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