Difference between revisions of "Team:Bielefeld-CeBiTec/Description"

 
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  <h1>Our Project</h1>    
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  <p>Here you find a description of our project.</p>
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<h1 style="margin-bottom: 0px">Foundations</h1>
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<p>How we built upon previous iGEM projects.</p>
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<p>The aim of our project was to make biosensors applicable in everyday life. Therefore, we developed two cell-free systems and tested several biosensors <i>in vivo</i> and <i>in vitro</i>.</p>
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<p>A central aspect was the establishment of a cell-free protein synthesis system. An ideal reporter protein is superfolder GFP (sfGFP). Consequently, we used sfGFP as a reporter gene and as a positive control for all our experiments. We started with the BioBrick <a href="http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a> by team Cambridge 2008 and observed that it was expressed in our CFPS. However, the expression strength was not sufficient for our biosensors. Therefore, we inserted a translation enhancing untranslated region (UTR) between the T7 promoter and the coding sequence, creating <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1758102">Part:BBa_K1758102</a>. We observed a drastic increase in fluorescence when testing this modified construct and performed our further experiments with this optimized sfGFP. We also tested whether the optimized UTR has an effect <i>in vivo</i> as well. We observed that the fluorescence of <i>E. coli</i> expressing the optimized sfGFP was significantly higher than the fluorescence of cultures expressing <a href="http://parts.igem.org/Part:BBa_I746909">BBa_I746909</a>. The difference could even be seen with the naked eye.</p>
  
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<h2> The Problem </h2>
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<p>The next step was to test biosensors for their functionality in our cell-free systems. Therefore, we used the following biosensors by previous iGEM teams:</p>
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        <li>arsenic (<a href="https://2006.igem.org/wiki/index.php/University_of_Edinburgh_2006" target="_blank">Edinburgh 2006</a>, <a href="http://parts.igem.org/Part:BBa_J33201" target="_blank">BBa_J33201</a>,  <a href="https://2013.igem.org/Team:Buenos_Aires" target="_blank">Buenos Aires 2013</a>, <a href="http://parts.igem.org/Part:BBa_K1106003" target="_blank">BBa_K1106003</a>)</li>
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        <li>mercury (<a href="https://2010.igem.org/Team:Peking" target="_blank">Peking 2010</a>, <a href="http://parts.igem.org/Part:BBa_K346002" target="_blank">BBa_K346002</a>)</li>
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        <li>nickel (<a href="https://2011.igem.org/Team:LMU-Munich" target="_blank">LMU Munich 2011</a>, <a href="http://parts.igem.org/Part:BBa_K549001" target="_blank">BBa_K549001</a>)</li>     
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        <li>lead (<a href="https://2007.igem.org/wiki/index.php/Lead" target="_blank">Brown 2007</a>, <a href="http://parts.igem.org/Part:BBa_I721003" target="_blank">BBa_I721003</a>)</li>
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        <li>chromium (<a href="https://2013.igem.org/Team:BIT/Project" target="_blank">BIT 2013</a>, <a href="http://parts.igem.org/Part:BBa_K1058007" target="_blank">BBa_K1058007</a>)</li>
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    <p>We improved the characterization of these biosensors by performing tests <i>in vivo</i> as well as in our CFPS. In particular, we improved the chromium sensor by <a href="http://parts.igem.org/Part:BBa_K1758313" target="_blank">codon-optimization</a>.</p>
  
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<p>Drinking water is the most basic of all resources, but its global exposure to toxic substances is a big problem. In particular, heavy metals endanger the health of millions of people. These metals occur naturally in small amounts in the earth’s crust. However, by erosion or human activities, such as mining or agricultures, they are released and can accumulate in soils and waters. That is why the groundwater in many regions of the world is contaminated for example with arsenic. Among others India and Bangladesh are particularly affected by this issue. But even in countries where the supply with clean water is a minor problem, heavy metal contaminations may occur as well, for example caused by old lead pipes. Since these last meters of water transport normally are not controlled, such contaminations often remain undetected for a quite long time. </br></br>
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    <p>Both of our biosensor designs make use of fluorescence as an output signal because this requires no cofactors or subtrates and offers a good sensitivity. However, fluorescence can usually not be seen with the naked eye. Nevertheless, it is possible to easily visualize fluorescence using a smartphone and filters. In 2014, <a href="https://2014.igem.org/Team:Aachen" taget="_blank">iGEM Aachen</a> have demonstrated this possibility using a filter for excitation. We improved this idea with a second filter in front of the camera, which enables everyone to specifally detect fluorescence by taking an image with their smartphone.</p>
  
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While some heavy metals are essential as trace elements, others lead to health consequences already in low concentrations. But in any case, the intake of larger amounts of heavy metals entails a variety of serious diseases such as nerve damage, cancer and even can lead to death. Therefore it is essential to control the heavy metal content of water and food. To enable this, a simple favorable and at the same time secure analytics is required. As laboratory tests and conventional test kits are in general not suitable for the public, we decided to establish a biosensor for various heavy metals.</br></br>
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<p>In summary, our project builds  upon the projects of several previous iGEM teams. We managed to improve the function and characterization of several of their BioBricks.</p>
  
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The second topic we want to deal with is motivated by the fact that recently crimes in which so-called knock-out-drugs (date-rape-drugs) were used become more frequent in our region.
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These drugs are usually a mixture of different substances which produce narcotic effects and thus make the victim defenseless and unable to act. By now the detection of knock-out-drugs is only possible in retrospect in the laboratory and this just in a limited time frame. However there is no opportunity for checking suspicious drinks quickly and easily. We want to change this by developing a biosensor for the detection of knock-out-drugs.</br>
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<a type="button" class="btn btn-default btn-next" href="https://2015.igem.org/Team:Bielefeld-CeBiTec/Project/Detection"><img src="https://static.igem.org/mediawiki/2015/c/cb/Bielefeld-CeBiTec_App_transparent.png"><p>Learn more about detecting fluorescence</p></a>
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<h2> The Detection System </h2>
 
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<p>Many organisms have genes that make them resistant to toxic substances or enable the use of particular nutrients. However, these genes are only required if the relevant substance is actually available. A permanent expression would be an unnecessary waste of energy for the organism. Therefore, control mechanisms have evolved ensuring the identification of specific substances to allow expression of certain genes only in presence of this substance. </br></br>
 
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The lac-operon which is required for the metabolism of lactose in Escherichia coli and many other bacteria is a classic example for such a mechanism. In absence of lactose, a repressor protein binds to the operator site of the lac-operon thereby obstructing the binding of the RNA polymerase to the promoter. Consequently there is no mRNA transcript of the following lac-genes. As the repressor is an allosteric protein it may assume two different shapes. In presence of lactose the inducer allolactose binds to the repressor triggering a conformational change. As a result the repressor releases the operator DNA and thus allows the RNA polymerase to bind to the promotor and express the genes.</br></br>
 
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In some bacteria similar systems enable the resistance to certain heavy metals. For instance there are bacteria which are able to grow in presence of arsenic, because they have a mechanism for carrying the heavy metal out of the cell. The required pump for this process is only formed when a specific repressor was previously inactivated by the binding of arsenic.</br></br>
 
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These natural mechanisms can be used to construct biosensors. Therefore, the genes that are naturally activated in the presence of a particular substance are replaced by a reporter gene. If this reporter gene is expressed, it provides a measurable signal. This signal may be, for example, a color change or a fluorescence signal.</br></br>
 
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Such biosensors allow a highly specific detection of minute amounts of a substance. Moreover, biosensors can be produced inexpensively whereby they are in particular of interest to developing countries.</br>
 
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<h2> Our Biosensor </h2>
 
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<p>Although, researchers are already working for many years on biosensors, these sensors have not yet arrived in everyday life. This can be explained with the fact that sensors based on living microorganisms create a number of problems. This involves on the one hand legal and security issues - since working with genetically modified organisms, their release into the environment must be prevented. On the other hand, these sensors are often not very user-friendly, as living cells have a limited suitability for storage and the application outside the laboratory is complicated. In order to solve these problems and thus make biosensors applicable in everyday life, we are working on two cell free systems for our biosensor.</br></br>
 
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Our first concept is based on the in vitro transcription and translation including protein biosynthesis outside of a living organism. It is already known that there is the possibility to run this central process in a cell extract. For this purpose the cells are disrupted and an energy-source as well as other supplements is added. This concept of cell-free protein synthesis (CFPS) is beneficial for example when a protein has to be produced which is toxic for the living cell.</br></br>
 
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Recently, it has been shown that it is possible to make the cell extract storable for a long time by freeze-drying it on paper (Pardee et al., 2014). With this freeze-dried cell extract protein synthesis was even possible after a long-term storage at room temperature only by adding water. In our opinion, this system is very promising for the development of easy-to-handle biosensors. Currently, we are working on the production of a cell extract and we intend to develop a test strip for heavy metals and knock-out drugs.</br></br>
 
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In parallel, we are working on a second concept that works with isolated proteins and DNA. This system is based on the interaction between a repressor protein and DNA. Thereby the repressor is immobilized on a surface and binds a small DNA molecule that carries its recognition sequence. Now, adding a water sample that contains the corresponding inducer causes a loss of the repressor-DNA-interaction and the DNA becomes detached. If this detaching is combined with the loss or generation of a signal, it is possible to detect whether the sample contains a certain substance or not.</br>
 
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The advantages of this system are its simplicity and thus its potential robustness. Furthermore, it works completely cell free and is therefore very interesting regarding biosafety.</br>
 
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<h2> Project Description </h2>
 
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<p>Tell us about your project, describe what moves you and why this is something important for your team.</p>
 
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We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be consist, accurate and unambiguous in your achievements.
 
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Judges like to read your wiki and know exactly what you have achieved. This is how you should think about these sections; from the point of view of the judge evaluating you at the end of the year.
 
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you though about your project and what works inspired you.</p>
 
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<p>See how other teams have described and presented their projects: </p>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Project"> Imperial</a></li>
 
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<li><a href="https://2014.igem.org/Team:UC_Davis/Project_Overview"> UC Davis</a></li>
 
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Latest revision as of 14:58, 29 October 2015

iGEM Bielefeld 2015


Foundations

How we built upon previous iGEM projects.

The aim of our project was to make biosensors applicable in everyday life. Therefore, we developed two cell-free systems and tested several biosensors in vivo and in vitro.

A central aspect was the establishment of a cell-free protein synthesis system. An ideal reporter protein is superfolder GFP (sfGFP). Consequently, we used sfGFP as a reporter gene and as a positive control for all our experiments. We started with the BioBrick BBa_I746909 by team Cambridge 2008 and observed that it was expressed in our CFPS. However, the expression strength was not sufficient for our biosensors. Therefore, we inserted a translation enhancing untranslated region (UTR) between the T7 promoter and the coding sequence, creating Part:BBa_K1758102. We observed a drastic increase in fluorescence when testing this modified construct and performed our further experiments with this optimized sfGFP. We also tested whether the optimized UTR has an effect in vivo as well. We observed that the fluorescence of E. coli expressing the optimized sfGFP was significantly higher than the fluorescence of cultures expressing BBa_I746909. The difference could even be seen with the naked eye.

The next step was to test biosensors for their functionality in our cell-free systems. Therefore, we used the following biosensors by previous iGEM teams:

We improved the characterization of these biosensors by performing tests in vivo as well as in our CFPS. In particular, we improved the chromium sensor by codon-optimization.

Both of our biosensor designs make use of fluorescence as an output signal because this requires no cofactors or subtrates and offers a good sensitivity. However, fluorescence can usually not be seen with the naked eye. Nevertheless, it is possible to easily visualize fluorescence using a smartphone and filters. In 2014, iGEM Aachen have demonstrated this possibility using a filter for excitation. We improved this idea with a second filter in front of the camera, which enables everyone to specifally detect fluorescence by taking an image with their smartphone.

In summary, our project builds upon the projects of several previous iGEM teams. We managed to improve the function and characterization of several of their BioBricks.

Learn more about detecting fluorescence