Difference between revisions of "IGEM:Imperial/2010/Experiments1"

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(New page: =Experiments for the Output Amplification Model= * Determine maximum concentration of sD that cells can produce – crucial! (''in vivo''): Compare activities with the next one * Determine...)
 
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=Experiments for the Output Amplification Model=
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* Determine maximum concentration of sD that cells can produce – crucial! (''in vivo''):
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<h1>Experiments for the Output Amplification Model</h1>
Compare activities with the next one
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* Determine how much XylE we need to produce prior adding catechol (the threshold value to be determined). (''in vitro then maybe in vivo'')
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<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/4/4f/Postit_large.jpg/500px-Postit_large.jpg"><div style="position:relative;top:-450px;left:50px;font: 11pt helvetica, georgia, courier, arial; margin-left: 10px;"><b>Output Amplification Model</b><br /><br />
In vivo IPTG? -> compare the cultures that have less of it
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<ol>
* Determine XylE and TEV production. (''in vivo'')
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<li>Determine the maximum concentration of sD that <br />cells can produce (in vivo):
XylE -> maybe, but very noisy. Use robot to induce production and measure activities
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Compare activity with 2.
TEV -> FRET pairs with TEV link
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</li>
* Degradation of XylE (''in vivo'' or ''in vitro'' if the cell division is not taken into account)
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<li>Determine how much XylE we need to produce <br />prior to adding Catechol (in vitro first, maybe in vivo <br />afterwards). In vivo IPTG: Compare cultures that <br />have less of it.
Yes -> Monitoring activity and then approximate the concentration. Remove the IPTG. Use robot
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</li>
* Can we determine TEV kinetics (kcat, Km?) on XylE-GFP? (''in vitro'')
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<li>Determine XylE and TEV production (in vivo). <br />Use robot to induce production and measure <br />activities TEV (FRET pairs with TEV link).
Yes
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</li>
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<li>Degradation of XylE (in vivo or in vitro if cell division <br />is not taken into account). Monitoring activity, then <br />approximate the concentration. Remove the IPTG.  
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</li>
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<li>Determine TEV kinetics (k<sub>cat</sub>, K<sub>m</sub>) on <br />XylE-GFP (in vitro).</li>
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</ol>
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</div>
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Revision as of 07:00, 9 September 2010

<html> <h1>Experiments for the Output Amplification Model</h1>

<img src="http://upload.wikimedia.org/wikipedia/commons/thumb/4/4f/Postit_large.jpg/500px-Postit_large.jpg"><div style="position:relative;top:-450px;left:50px;font: 11pt helvetica, georgia, courier, arial; margin-left: 10px;"><b>Output Amplification Model</b><br /><br /> <ol> <li>Determine the maximum concentration of sD that <br />cells can produce (in vivo): Compare activity with 2. </li> <li>Determine how much XylE we need to produce <br />prior to adding Catechol (in vitro first, maybe in vivo <br />afterwards). In vivo IPTG: Compare cultures that <br />have less of it. </li> <li>Determine XylE and TEV production (in vivo). <br />Use robot to induce production and measure <br />activities TEV (FRET pairs with TEV link). </li> <li>Degradation of XylE (in vivo or in vitro if cell division <br />is not taken into account). Monitoring activity, then <br />approximate the concentration. Remove the IPTG. </li> <li>Determine TEV kinetics (k<sub>cat</sub>, K<sub>m</sub>) on <br />XylE-GFP (in vitro).</li> </ol> </div> </html>