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From 2011.igem.org

Method

We created two following constructions to measure gene expression depending on the concentration of glutamine. We used BBa_J23101 as a promoter for construction1,which is used as the criteria, and binding sites and σ-54 promoter for construction2, which is object to be measured.

We also measured the amount of mRNA to calculate RPU of the steady state for the each concentration of glutamine. First, we made following preliminary experiment to measure the length of times before steady state.

We cultivated E.coli in M9 media(+ casamino acid) for about 15 hours and dispenced 2.4ml to each tube. Then, we centrifuged these tubes (13,000 rpm , 4℃, 1min) and discarded the supernatant. We added 1.2ml media(- casamino acid) and centrifuged at 4℃ twice. Again, we centrifuged these tubes and discarded the supernatant and added 1.2ml media(-casamino acid) at 37 ℃. We brought up E.coli at 0,5,10,15,20,25,30,60min and extracted RNA and synthesized cDNA. Finally, we used real time PCR.

RPU is defined as follows.