Difference between revisions of "Team:Tuebingen/Description"

 
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<h2> Project Description </h2>
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<h2>A Biosensor Memory Module: Cre Sensor</h2>
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<p>Analysis and imaging methods are an indispensable element of every scientific study - in a wider sense the results are only as good as their measurement methods are.
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Photostimulation is one of the most important non-invasive analysis methods existing which allows researches to examine the relationship between metabolic processes, e.g. through activating a molecule via light treatment. What we want to do is to create a module in which we can do a snapshot of the activity of a sensor at any time.</p>
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<p>We want to design a module, which is capable to make a ‘snapshot’ of sensor activity at any time, which in turn activates a promotor and changes gene expression. This snapshot is induced by a certain wavelength.</br>
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For caging we use Dronpa, a fluorescent protein. Dronpa is able to dimerise at 400nm and monomerises again at 500nm. Therefore it can be used reversibly for caging an enzyme. If a amino- and carboxy-terminus bound Dronpa dimerises, it will cover the active site of the enzyme and inactivates it this way. One can control this reversible mechanism by light.</p>
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<p>To proof a successful activation of the enzyme a reporter is necessary. The Cre-recombinase suits well for this. Cre can be inserted into the DNA permanently as a sensor and is able to be controlled by light if bound to Dronpa. When activated, it can lead to the expression of a GFP gene construct, which is easily proofable due to its fluorescence.</br>
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This module can be used with a variety of associated enzymes and proteins to light-dependently controll these. The possible applications are plenty and the augur analytical and diagnostical value.</p>
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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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<h5>What should this page contain?</h5>
 
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<li> A clear and concise description of your project.</li>
 
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<li>A detailed explanation of why your team chose to work on this particular project.</li>
 
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<li>References and sources to document your research.</li>
 
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<h4>Advice on writing your Project Description</h4>
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<p xmlns="http://www.w3.org/1999/xhtml" style="font-size:46px;font-weight:bold;color:black;">The expression of the Dronpa-Cre-Dronpa construct is under control of a promotor, that is influenced by a sensor.
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Any biosensor that is able to positively regulate a promotor can be used in our setting.</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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<p xmlns="http://www.w3.org/1999/xhtml" style="font-size:52px;font-weight:bold;color:black;">After expression the Dronpa-Cre-Dronpa construct is constituted of fluorescent Dronpa domains that attach to each other and thereby inhibit the Cre domain sterically.</p>
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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 xmlns="http://www.w3.org/1999/xhtml" style="font-size:52px;font-weight:bold;color:black;">Illumination with violet light (400nm) re-activates Dronpa fluorescence and also leads to the closed conformation of the construct.</p>
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<p xmlns="http://www.w3.org/1999/xhtml" style="font-size:47px;font-weight:bold;color:black;">Illumination with blue light (500nm) deactivates Dronpa fluorescence and thereby also multimerisation of the Dronpa domains.</p>
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<h4>References</h4>
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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 xmlns="http://www.w3.org/1999/xhtml" style="font-size:52px;font-weight:bold;color:black;">In the induced open conformation of the construct, the Dronpa domains are only losely connected to the Cre, which thereby becomes active.</p>
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<p xmlns="http://www.w3.org/1999/xhtml" style="font-size:46px;font-weight:bold;color:black;">The RFP-luciferase reporter switch leads to expression of RFP if the memory system has not been activated by CREllumination.</p>
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<p xmlns="http://www.w3.org/1999/xhtml" style="font-size:52px;font-weight:bold;color:black;">After activation of the Cre protein it removes the loxp-RFP-loxp part of the reporter switch. This leads to expression of luciferase, which serves as the final reporter of the system.</p>
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<h2> Project Description</h2>
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<h4> A Biosensor Memory Module: Cre Sensor</h4>
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<div style="text-align: justify; margin-right: 10px;">
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<p><Analysis and imaging methods are an indispensable element of every scientific study - in a wider sense the results are only as good as their measurement methods.<br/>
 +
Photostimulation is one of the most important non-invasive analysis methods allowing researchers to examine the relationship between metabolic processes, e.g. through activating a molecule via light treatment. We want to create a module which makes it possible to take a snapshot of the activity of a sensor at any time.
 +
Our designed system should be capable of capturing such a ‘snapshot’ of the activity of a sensor. Therefore our aim is to create a Cre recombinase whose activity can be reversibly controlled by light. By activating this construct only for a short period of time, we can use a Cre to switch on expression of Luciferase in only a limited amount of the sensor cells. Through coupling the Cre expression to the sensor we can thereby permanently write the sensor state of a given time point into the DNA of a system. </p>
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<p>
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To achieve the construction of a reversibly activatable Cre recombinase we want to apply the caging mechanism described by Zhou et al <a href="https://2015.igem.org/Team:Tuebingen/References">[Zhou 2012]</a>. This caging is performed by fusing a copy of a variant of the fluorescent protein Dronpa to both the C- and N-terminus of the Cre recombinase. Since this Dronpa variant is able to form monomers or dimers depending on illumination with light of different wavelengths, we hope that the dimerized form inhibits the activity of the Cre recombinase. </p>
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<p>
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Because our system only needs the expression of the caged Cre construct to be dependent on a sensor, it can be combined with almost all Biosensors that include a means of transcriptional control. This gives the system a wide variety of possible applications, especially in the context of the work of other iGEM teams.</p>
  
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<h4>Inspiration</h4>
 
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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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<li><a href="https://2014.igem.org/Team:SYSU-Software/Overview">SYSU Software</a></li>
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Latest revision as of 14:57, 9 November 2015

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Project Description

A Biosensor Memory Module: Cre Sensor

Photostimulation is one of the most important non-invasive analysis methods allowing researchers to examine the relationship between metabolic processes, e.g. through activating a molecule via light treatment. We want to create a module which makes it possible to take a snapshot of the activity of a sensor at any time. Our designed system should be capable of capturing such a ‘snapshot’ of the activity of a sensor. Therefore our aim is to create a Cre recombinase whose activity can be reversibly controlled by light. By activating this construct only for a short period of time, we can use a Cre to switch on expression of Luciferase in only a limited amount of the sensor cells. Through coupling the Cre expression to the sensor we can thereby permanently write the sensor state of a given time point into the DNA of a system.

To achieve the construction of a reversibly activatable Cre recombinase we want to apply the caging mechanism described by Zhou et al [Zhou 2012]. This caging is performed by fusing a copy of a variant of the fluorescent protein Dronpa to both the C- and N-terminus of the Cre recombinase. Since this Dronpa variant is able to form monomers or dimers depending on illumination with light of different wavelengths, we hope that the dimerized form inhibits the activity of the Cre recombinase.

Because our system only needs the expression of the caged Cre construct to be dependent on a sensor, it can be combined with almost all Biosensors that include a means of transcriptional control. This gives the system a wide variety of possible applications, especially in the context of the work of other iGEM teams.