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<p><h1>Further Characterization of Kill Switch (<a href="http://parts.igem.org/Part:BBa_K2086002">K2086002</a>)</h1></p> | <p><h1>Further Characterization of Kill Switch (<a href="http://parts.igem.org/Part:BBa_K2086002">K2086002</a>)</h1></p> | ||
<font color="silver">Due to time constraints, after the summer ended, complete characterization of our nitrate-sensitive kill switch was unable to be accomplished. However, we were able to qualitatively assess its viability as a kill switch. The BioBrick was ligated into a lower copy-count plasmid, <a href="http://parts.igem.org/Part:pSB4C5">pSB4C5</a>. Competent cells of our auxotrophic ΔserA strain were then transformed with the low copy-count kill-switch. These were then subcultured cultured into two samples of minimal media: one with no nitrate ions, and one with 4mM NO3<sup>-</sup> and were grown anaerobically overnight. As seen in the image below, the sample induced with nitrate was able to grow, while the media lacking nitrates had no discernible growth.<sup>[A]</sup> | <font color="silver">Due to time constraints, after the summer ended, complete characterization of our nitrate-sensitive kill switch was unable to be accomplished. However, we were able to qualitatively assess its viability as a kill switch. The BioBrick was ligated into a lower copy-count plasmid, <a href="http://parts.igem.org/Part:pSB4C5">pSB4C5</a>. Competent cells of our auxotrophic ΔserA strain were then transformed with the low copy-count kill-switch. These were then subcultured cultured into two samples of minimal media: one with no nitrate ions, and one with 4mM NO3<sup>-</sup> and were grown anaerobically overnight. As seen in the image below, the sample induced with nitrate was able to grow, while the media lacking nitrates had no discernible growth.<sup>[A]</sup> | ||
+ | |||
+ | [A] <a href="https://2016.igem.org/Team:UNebraska-Lincoln/Attributions">Attributions</a> | ||
</section> | </section> |
Revision as of 06:34, 19 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 (K2086002). 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.