Team:UNebraska-Lincoln/experiments

<!DOCTYPE HTML> Elements - Solid State by HTML5 UP

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 a ΔserA strain of competent cells with our BioBrick BBa_K2086002. Growing these cells in minimal media resulted in growth whereas un-complemented cells would be unable to grow due to the lack of serine.