Difference between revisions of "Part:BBa J37015:Experience"

Latest revision as of 12:17, 26 September 2012

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Applications of BBa_J37015

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UNIQ6f825394f9634730-partinfo-00000000-QINU UNIQ6f825394f9634730-partinfo-00000001-QINU This part was tested by the Brown iGEM team in August 2007. Some interesting and very repeatable results were obtained. It was expected that this part, J37015, would produce significantly more GFP than the part T9002, which differs only in that it has no LuxI gene and therefore no positive feedback loop. It was also expected that higher amounts of AHL would cause J37015 to produce more GFP than low amounts of AHL. Both of these hypotheses were proven false.

In this experiment, 10 uL of a solution of AHL in water was mixed with 90 uL of LB and 3 uL of an overnight culture of cells. The concentrations 0, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 uM, 10 uM, 100 uM, and 1 mM AHL were tested. These are the final concentrations of AHL in the solution with LB and cells, not the concentration of AHL in the water that was added to that solution. Measurements were made using a NanoDrop Spectrophotometer for Cell Density and a NanoDrop Flourospectrometer for GFP fluorescence at several time points over a 24 hour period. The average of all GFP measurements at the first measurement, time 0, was considered background fluorescence, and was subtracted from all subsequent GFP measurements. Additionally, all GFP measurements were divided by the cell density so that GFP per cell could be measured. This was to prevent noise from potential effects of AHL concentration on cell growth.

The first graph shows GFP output at 5, 7, and 24 hours after addition of AHL to the cells for each of the concentrations listed above. This clearly shows an inverse relationship between concentration of AHL and GFP output. This is quite surprising, because it was expected that more AHL would lead to more GFP expression. We do not know why this occurs, and are studying it further. Also, the fact that there is so much GFP expression when no AHL is added to the system indicates that this promoter acts at some constitutive level, and therefore has no use in a signal amplification system because it will always produce a signal regardless of its input.

The second graph shows the same result as the first, but looks only at the 5 hour time point. All time points after the first 2 hours showed indicated the inverse relationship between AHL concentration and GFP production described above, but it is most clear at 5 hours.

The third graph shows a different result. The same concentration of AHL, 1 mM, was added to both J37015 and T9002. The only difference between these parts is that J37015 has the LuxI gene between its pLux promoter and its GFP gene. This theoretically creates a positive feedback loop, which should drive the cell to produce as much GFP as possible when it encounters any AHL. However, T9002 clearly had far more GFP output than J37015. This is even more intriguing when you consider the fact that T9002 has a weaker ribosome binding site for GFP than J37015 by 3 fold. One possible explanation for this is that the GFP gene is further from the promoter in J37015 than in T9002 due to the insertion of LuxI. However, it seems unlikely that this could cause such a strong difference.

From these results, it appears that J37015 does seem to have some sort of function, though not as a signal amplification device. Further investigation will hopefully reveal the cause of these unexpected data.

Thanks a lot for Brown Team's contribution! I am a member of Tsinghua Team 2012. We get this part from kit 2012 but it doesn't work at all. We digest the part using EcoR1 and Pst1 and the band of part shows that it is only about 1kb. That is very strange. Then we sequence the part and we find that the sequence is totally wrong. We don't know what's that. The team Brown has tested this part in 2007 so that we think there is something wrong with the kit in the following years.

UNIQ6f825394f9634730-partinfo-00000002-QINU