IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1

Results summary

pT7-GFP In Vitro

37 Degrees

The pT7 was tested in vitro for a span of 4 hours at 37^oC right after iPTG induction. After the initial reading, it was found that fluorescence decreased down to a steady level. This was observed for all our 3 samples and negative control, indicating that it was due to a change in the in vitro background fluorescence. The possible source of this decrease could be due to an extra experimental step taken, which was a quick centrifugation before the plate was read in the fluorometer.

Results of in vitro testing of pT7 over a 4 hour period at 37^oC

The graph on the right dispays the following.

Fluorescence of the diluted GFP solution. (+ve control)

The average fluorescence of the pT7 samples(3)

The fluoresence of a solution containing only S30 cell extract (-ve control).

As you can observe, the pT7 does not appear to be working in vitro either. The commercial S30 cell extract used does not promote it to start expressing GFP at least within the 4 hours during which our tests were carried out and its fluorescene levels remain well below the diluted GFP.

The plate containing the samples was stored in a 37^oC incubator overnight. It was re-tested the next morning to see whether GFP had been expressed,22 hours after of induction. The results were joined with the initial testing done over the first 4 hours of induction and are shown below.

Results of in vitro testing of pT7 over a 29 hour period at 37^oC

The graph legend is the same as the one of the graph above. The fluorescence the next day (after 22 hours of induction) had risen a bit but so did our -ve control. This leads us to suspect that some of our samples had been contaminated perhaps with GFP from the +ve control. From this we realised we had to re-think the way our samples were arranged on the well plates. We had to allow more spacing betweeen the samples and avoid placing samples next to adjacent wells.

Overall though, it can be concluded that the pT7 construct does not work in vitro.

Experiment 1

• Concentration of pTet-GFP = 110ng/μl
• Concentration of pT7-GFP = 100ng/μl
• Concentration of pCI-GFP =

Experiment 2

pTet worked in vitro for about 3 hours. (The fluorescence reading for pTet in E.coli commercial cell extract was found to be increasing until the third hour.)
pT7 was not tested as it was proven to work in the In Vivo test.

pT7-GFP

• 37^oC - The pTet was tested in vitro for a span of 4 hours at 37^oC. After the initial reading, it was found that fluorescence decreased down to a steady level. This was observed in our samples and negative control, indicating that it was due to a change in the in vitro background fluorescence. The reason for this decrease could be due to an extra experimental step, which was a quick centrifugation before the plate was read in the fluorometer.

Results of testing pT7 in vitro at 37^oC at 0.15 sec counting time for fluorometer detector

Complete set of results and raw data