IGEM:IMPERIAL/2007/Experimental Design/Phase2/Results 2.2.3

Results Summary

pTet-LuxR-pLux-GFP

After testing a range of 1nM,10nM,50nM,100nM,1000nM, we have now defined the range of AHL concentration that we are interested in is 1-10nM. This test was for 3nM,5nM,7nM.

The results show us that the threshold of detection of AHL is at least 3nM, given this we are now going to repeat tests with lower AHL concentrations. In addition the results show is that the maximum output of fluorescence decreases with decreasing AHL concentrations. This agrees with our knowledge from the characterisation of the BBa_F2620 construct.

However, comparison with our previous AHL concentrations at 10nM, 50nM, 100nM, the maximum output of fluorescence does not correlate. The maximum output from the 100nM fluorescence was around 40,000. The reason is thought to be down to two causes:

1. The cell extract bought from promega was a different batch and so may have had variation
2. The DNA prep was different, the for the first testing a midi prep was used and for the second testing a maxi prep was used. Although we carried out dilutions to give the same overall concentration, this may not have been enough to prevent variation. The small concentration of the mini prep may have made the A260 reading unreliable or the salt concentration may have varied.

From this testing we are now going to test further concentrations between 1 and 10nM. In addition our methodology is going to change, we are now going to use the same maxi prep throughout our testing and try to keep to the same batch of cell extract. We will also research into how much variation there is in the Promega S30 cell extract.

Complete set of results and raw data