"
Team:Carnegie Mellon/InterLab
From 2014.igem.org
Interlab Study
Goal: To collect fluorescence data from three devices from different teams around the world
Study Description and Plasmids
Two plasmids were constructed using parts given to us from the iGEM registry. The first contained a high expressing, constitutive J23101 promoter with GFP, while the second one contained a very low-expressing, constitutive J23115 promoter with GFP. The third plasmid was already constructed, and contained the same promoter as the first plasmid, however, it was contained in a low-copy plasmid - pSB3K3.
We constructed these devices by digesting these three plasmids, the J23101 promoter, the J23115 promoter and the E0240 (GFP), with restriction enzymes XbaI and SpeI. We then ligated the GFP fragment into the plasmids containing the promoters. We then transformed these plasmids back into E. coli MACH cells to screen the cells for fluorescence. After identifying the cells containing both the promoter and the GFP, we streaked these colonies out and then repeated the selection for cells containing GFP. The DNA from overnight cultures made of these cells was extracted, then transformed these two plasmas, and the third pSB3K3 plasmid (containing the J23101 promoter and GFP) into competent Top10 E. coli cells. We then measured fluorescence from these samples in triplicate (along with non-expressing control cells), using the TECAN, to get fluorescence values in RFU (relative fluorescent units).
Results
Conclusions
From the TECAN reader results we concluded that the fluorescence of the cells corresponded to the type of promoter - with J23100 having the highest expression with a signal to noise ratio of 22,365, and the low copy plasmid pSB3K3 with the same promoter having the second highest fluorescence, with a signal to noise ratio of 3,183. The low expressing J23115 promoter had the lowest expression, with a signal to noise ratio of 140.
