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

In Vitro Tetsing

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Aim

The aims of this experiment is to test the following constructs in vitro at 37°C To Determine if the following constructs work in vivo:

• pTet-GFP
• pT7-GFP

After this testing we will test working constructs at 10°C and 37°C. In addition we want to identify any problems with our testing method

Both constructs were tested in vitro on *pTet and pT7 in vitro Tested 21-08-2007 to Tested 23-08-2007

pTet was tested in vitro at 10°C and 45°C Tested 21-08-2007

Results

pTet-GFP(100ng/μl)

Test: 21-08-2007

37 Degrees

17-08-2007- Results of pTet and pT7

We tested pTet-GFP in vitro in commercial S30 Cell Extract at 37 °C. The initial test was carried out over a period of 6 hours, during which measurements were taken every hour. The positive control of GFP diluted in water shows a typical expotential decay. The negative control remains relativly constant, however it does increase over the duration of 4 hours. The Graph to the left shows the following data: Fluorescence of the GFP diluted in water solution (+ve control to show if fluoreometer is set correctly for GFP) The average fluorescence of the pTet samples(3) The fluoresence of a solution containing only S30 cell extract (-ve control).

Test: 21-08-2007 to Test: 23-08-2007

37 Degrees

Results of in vitro testing of pTet over a 56 hour period

The pTet-GFP in vitro samples were left and restested over three days to test to see what a typical expression curve looks like. The results of the average fluorescence of the samples and the positive control are shown to the left. The graph on the left shows the following data: Fluorescence of the diluted GFP solution. (+ve control) The average fluorescence of the pTet samples(3)

Test: 22-08-2007

10 Degrees

22-08-2007- Results of pTet at 10°C

We tested pTet-GFP in vitro in commercial S30 cell extract at 10°C for 4 hours, sampling every 30 minutes. The results show us that the pTet-GFP does not work at 10 °C. The fluorescence of the samples remains nearly constant to that of the negative control. However, the negative control and the sample show slight variation around the basal level. Again for 10°C the positive control shows an expotential decay, however again there is variation in the curve. The graph the the right corresponds to the following: Positive Control - Fluorescence of the diluted GFP solution The average fluorescence of the pTet samples(3) Negative Control - The fluoresence of a solution containing only S30 cell extract

Test: 22-08-2007

45 Degrees

22-08-2007- Results of pTet at 45°C

We tested pTet-GFP in vitro in commcercial S30 extract at 45°C for 4 hours, sampleing every 30minutes. The results show that there minimal expression of GFP at 45°C. The sample only slightly increases above the negative control showing minimal expression. The positive control again shows expotential decay. Interestingly there is even greater variation within the results, giving very unpreditable trend lines. The results to the left show the followig Positive Control - Fluorescence of the diluted GFP solution The average fluorescence of the pTet samples(3) Negative Control - The fluoresence of a solution containing only S30 cell extract

pT7-GFP In Vitro (100ng/μl)

37 Degrees

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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.

Complete set of results and raw data