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Team:Carnegie Mellon/InterLab
From 2014.igem.org
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<p><img src="https://static.igem.org/mediawiki/2014/d/d1/Interlab_Plasmids.png" alt="Plasmids"><p> | <p><img src="https://static.igem.org/mediawiki/2014/d/d1/Interlab_Plasmids.png" alt="Plasmids"><p> | ||
<p><b>Description:</b> 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 <i>E. coli</i> 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 <i>E. coli</i> 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).<p> | <p><b>Description:</b> 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 <i>E. coli</i> 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 <i>E. coli</i> 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).<p> | ||
| - | <p>< | + | <p><b>Results:</b><p> |
<hr> | <hr> | ||
Revision as of 05:38, 6 October 2014
Interlab Study
Goal: To collect fluorescence data from three devices from different teams around the world
Plasmids
Description: 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:
