"

From 2010.igem.org

Characterising the Biobrick

With our biobrick made, we then needed to establish whether it worked, and over what range. We began by checking simply whether it responded to nitrate at all. Then, having determined that it did, conducted a more detailed assay across the range of nitrate levels for which the biobrick is most sensitive.

Having established the biobrick was definitely switched on by 20mM of KNO3, we wanted to conduct a broader test of its behaviour across a range of different nitrate levels

Does it work? Yes!

To begin with we simply wanted to determine whether the biobrick worked as expected - producing GFP in the presence of nitrates, but not without.

Two 5mL overnights were set up, one without any nitrogen and one with 20mM of Potassium Nitrate KNO3, made up according to the table below. This level was chosen as previous work by the Edinburgh 2009 iGEM team had shown the promoter strongly active by this point.

Nitrate (mM)	LB (mL)	KNO3 (μL)
0	5	0
20	5	102

The cultures were then left in the 37°C shaker overnight and examined the next morning. We were pleased to note that the culture exposed to nitrate was visibly green whilst the one without was not, suggesting our new biobrick worked at least at the most basic ‘on/ off’ level.

An assay was set up between 0 – 10 mM of Potassium Nitrate at 2mM intervals. The same colony of MG1655 containing BBa_K381001 was used to inoculate six 5mL tubes of LB at varying nitrate levels as listed below. This was repeated twice to allow the results to be averaged.

Average:

Unfortunately, because we only ran two repeats, we cannot be certain whether the downward curve of the graph is true behaviour or the result of one the 10mM readings being extremely low