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Team:Carnegie Mellon/InterLab
From 2014.igem.org
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<p>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>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><h2>Results</h2><p> | <p><h2>Results</h2><p> | ||
| - | <p><img src="https://static.igem.org/mediawiki/2014/ | + | <p><img src="https://static.igem.org/mediawiki/2014/f/f7/Interlab_Data_.png"></p> |
<p><img src="https://static.igem.org/mediawiki/2014/e/ee/Interlab_data_2.png" alt="interlab data 2"</p> | <p><img src="https://static.igem.org/mediawiki/2014/e/ee/Interlab_data_2.png" alt="interlab data 2"</p> | ||
<p><img src ="https://static.igem.org/mediawiki/2014/8/86/Interlab_Results.png" alt="interlab results"</p> | <p><img src ="https://static.igem.org/mediawiki/2014/8/86/Interlab_Results.png" alt="interlab results"</p> | ||
Revision as of 06:04, 6 October 2014
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
