Difference between revisions of "Team:UNebraska-Lincoln/experiments"

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<h2 class="major">Experiments and Results </h2>
 
<h2 class="major">Experiments and Results </h2>
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<p><h1>Recharacterization of PyeaR-GFP (<a href="http://parts.igem.org/Part:BBa_K381001">K381001</a>)</h1></p>
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<p><h1>Re-characterization of PyeaR-GFP (<a href="http://parts.igem.org/Part:BBa_K381001">K381001</a>)</h1></p>
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<font color="silver">Central to our project is our nitrate-sensitive kill switch, which relies on the Nitrate-sensitive yeaR promoter, originally submitted by the Edinburgh iGEM team in 2009 (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K216005">K216005</a>). In order to confirm the activity of this promoter, we recharacterized a composite PyeaR-GFP composite biobrick, assembled by the BCCS-Bristol iGEM team in 2010 (<a href="http://parts.igem.org/Part:BBa_K381001">K381001</a>). To do so we transformed competent genehogs cells with the plasmids provided in the iGEM Distribution Kit and then plated them and allowed them to grow overnight. The following day they were inoculated in 5mL of LB broth and again allowed to grow overnight. The culture of cells was then subcultured into a 96 well plate and the subcultures were then activated? With varying concentrations of Potassium? Nitrate. The subcultures were then allowed to grow further as to enable them to express the green fluorescence protein. Their expression of the GFP gene was then measured using a plate reader which allowed a quantitative determination as to the effectiveness of the yeaR promoter at varying nitrate levels. </font>
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<font color="silver">Central to our project is our nitrate-sensitive kill switch. Our kill switch relies on the Nitrate-sensitive yeaR promoter, originally submitted by the Edinburgh iGEM team in 2009 (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K216005">K216005</a>). In order to confirm the activity of this promoter, we re-characterized a composite PyeaR-GFP composite biobrick, assembled by the BCCS-Bristol iGEM team in 2010 (<a href="http://parts.igem.org/Part:BBa_K381001">K381001</a>). To do so we transformed competent genehogs cells with the plasmid provided in the iGEM Distribution Kit. The following day, the transformed cells were inoculated in 5mL of LB broth and again allowed to grow overnight. The culture of cells was then subcultured into a 96 well plate and the subcultures were induced with varying concentrations of potassium nitrate. The induced subcultures were then grown overnight to allow the GFP to be fully expressed. The next day, the fluorescence (rfu) of each subculture was collected in order to plot the activity (GFP expression) against concentration of nitrate ion. </p>
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<p> We also created a time-based curve (confirming past results from Bristol-BCCS) where fluorescence measurements (rfu) were recorded every half hour. We were then able to plot the activity against time, with varying concentration of nitrate ion.</font></p>  
  
 
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Revision as of 06:18, 18 October 2016

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Experiments and Results

Re-characterization of PyeaR-GFP (K381001)

Central to our project is our nitrate-sensitive kill switch. Our kill switch relies on the Nitrate-sensitive yeaR promoter, originally submitted by the Edinburgh iGEM team in 2009 (K216005). In order to confirm the activity of this promoter, we re-characterized a composite PyeaR-GFP composite biobrick, assembled by the BCCS-Bristol iGEM team in 2010 (K381001). To do so we transformed competent genehogs cells with the plasmid provided in the iGEM Distribution Kit. The following day, the transformed cells were inoculated in 5mL of LB broth and again allowed to grow overnight. The culture of cells was then subcultured into a 96 well plate and the subcultures were induced with varying concentrations of potassium nitrate. The induced subcultures were then grown overnight to allow the GFP to be fully expressed. The next day, the fluorescence (rfu) of each subculture was collected in order to plot the activity (GFP expression) against concentration of nitrate ion.

We also created a time-based curve (confirming past results from Bristol-BCCS) where fluorescence measurements (rfu) were recorded every half hour. We were then able to plot the activity against time, with varying concentration of nitrate ion.