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From 2010.igem.org

Characterising the Ratio Method

0 - 10mM Assay

Having made our double transformants the next step was to characterise their response to Nitrate. We began by examining this between 0 and 10mM of Potassium Nitrate (in 2mM steps). 5mL solutions of LB and Potassium Nitrate were made up in sterilins and inoculated with the same colony of MG1655s. These were then left overnight at 37°C and their fluorescence analysed using the photo-spectrometer the next morning. This gave results as follows (Where data is missing, colonies produced neither GFP nor RFP, thus were discounted as failures, note how this highlights the usefulness of the double transform method in stopping false negatives):

Nitrate (mM)	GFP	RFP	GFP / RFP
0	0.045	0.0205	0.45556
2	0.061	0.1057	1.73279
4	n/a	n/a	n/a
6	0.0522	0.1128	2.16092
8	n/a	n/a	n/a
10	0.0429	0.1185	2.76224

As is evident from the graph below [put nice graph here!] there is a clear trend of an increase in GFP/RFP with increasing Nitrate levels, indicating our double transformants had indeed worked.

0 - 2mM Assay

Having established our ratio method worked between 0 - 2mM KNO3, we were keen to check whether it worked at levels more relevant to our particular project. Consultation with farmers established typical Nitrate levels in soil lay began 0 - 2mM, thus we decided to characterise the double transformants further in this range.

As before, 5mL solutions of LB and Potassium Nitrate were set up, inoculated, and left overnight at 37°C. We then analysed their fluorescence using the photo-spectrometer, yielding the following results:

Nitrate (mM)	ratio test 1	ratio test 2	ratio test 3	average
0	n/a	0.70025	0.54227	0.62126
0.4	1.17228	n/a	1.10214	1.13721
0.8	n/a	1.34615	1.27687	1.31151
1.2	n/a	n/a	n/a	n/a
1.6	n/a	1.83442	1.61957	1.727
2.0	1.41866	n/a	2.414	1.91633

Again this allowed us to plot a graph indicating behaviour [insert graph!] that shows a clear upward trend in ratio with Nitrate, indicating our ratio method works well not only across a broad range, but also within the Nitrate levels that are important for this project. Further, this data allowed us to formulate an equation linking the fluorescence to Nitrate levels [insert equation!]