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 lab was closed between hours 4:00 and 22:00 and hence a large array of readings is missing. But we are only interested whether the construct works. With that in mind and given the slow degradation rate of GFP we should be able to detect if it was expressed even 20 hours later. 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, the fluorescence readings were minimal compared to pTet.It can thus be concluded that the pT7 construct does not work in vitro with the commercial S30 cell extract.

pTet-GFP

37 Degrees

The pTet-GP assembly was tested using commercial S30 cell extract to conclude that as opposed to pT7, it works. Unfortunately it does not work as eficiently as it does in vivo at the same temperature (37^oC). The initial test was carried out over a period of 6 hours, during which measurements were taken every hour. The results are shown below:

Results of in vitro testing of pTet over a 6 hour period

The graph on the right dispays the following.

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

The average fluorescence of the pTet samples(3)

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

In just approximately 1 hour after the DNA was added to the cell extract, GFP is already expressed. Even though we obtained a fluorescence level well above the negative control (near zero fluorescence) it showed only a slight increase over the 6 hours and was 4 times less than our positive GFP control.

The samples were left overnight and re-tested next morning (10:00AM) to see if there were any changes in the levels of expression. Readings were taken every hour for the second day of testing as well. Finally, the samples were retested again for a third day after they had been left for a second night in the incubator. The results from all 3 days (The initial 6-hour-Day 1 period, Day2 and Day3 )are shown below:

Results of in vitro testing of pTet over a 6 hour period

The graph on the right dispays the following.

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

The average fluorescence of the pTet samples(3)

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

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.

Complete set of results and raw data