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>
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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 />
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In vivo IPTG? -> compare the cultures that have less of it
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<ol>
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* 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):
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XylE -> maybe, but very noisy. Use robot to induce production and measure activities
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Compare activity with 2.
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TEV -> FRET pairs with TEV link
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</li>
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* 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.
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Yes -> Monitoring activity and then approximate the concentration. Remove the IPTG. Use robot
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</li>
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* 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).
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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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</html>

Revision as of 09:00, 9 September 2010

Experiments for the Output Amplification Model

Output Amplification Model

  1. Determine the maximum concentration of sD that
    cells can produce (in vivo): Compare activity with 2.
  2. Determine how much XylE we need to produce
    prior to adding Catechol (in vitro first, maybe in vivo
    afterwards). In vivo IPTG: Compare cultures that
    have less of it.
  3. Determine XylE and TEV production (in vivo).
    Use robot to induce production and measure
    activities TEV (FRET pairs with TEV link).
  4. Degradation of XylE (in vivo or in vitro if cell division
    is not taken into account). Monitoring activity, then
    approximate the concentration. Remove the IPTG.
  5. Determine TEV kinetics (kcat, Km) on
    XylE-GFP (in vitro).

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