Team:UNebraska-Lincoln/Results

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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.

Qualitative confirmation of kill Switch (K2086002) Activity

In order to verify that our kill-switch (K2086002) was able to successfully complement the strain of E. coli that we were using, we transformed competent ΔserA (E. coli JW2880) cells with our kill-switch biobrick. Growing these cells in minimal media resulted in growth whereas un-complemented cells were unable to grow due to the lack of serine.

Griess Reagent Standard Curve Creation

In order to assay the activity of our nitrate reductase gene (K2086000), we needed a way to be able to quantify the reduction of nitrate ions. The nitrate reduction can effectively be determined by the increase in concentration of the product, the nitrite ion. To do this, we used Griess Reagent, a common nitrite concentration indicator. Although time restrictions prevented us from being able to test the effectiveness of our BioBrick, we were able to construct the following standard curve by combining Griess Reagent with known concentrations of Sodium Nitrite in a 96 well plate and then using the plate reader to measure their absorbance at 540 nm.

Further Characterization of Kill Switch (K2086002)

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, pSB4C5. Competent cells of our auxotrophic ΔserA strain were then transformed with the low copy-count kill-switch. These were then cultured into two samples of minimal media: one with no nitrate ions, and one with 4mM NO3^- 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.^[A]

[A] Attributions

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