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

In vitro Testing of pTet-GFP and pT7-GFP Constructs

Aims

To Determine if the following constructs work in vitro:

• pTet-GFP
• pT7-GFP

To test the operating range of the constructs at 10°C, 37°C and 45°C over a staggered 24 hour period.

To identify problems in experimental methodology.

The testing was comprised of several tests:

• pTet and pT7 in vitro - Tested 21-08-2007 to 23-08-2007
• pTet in vitro at 10°C, 37°C and 45°C - Tested 21-08-2007

Materials and Methods

Refer to protocols page.

Results

pTet-GFP(100ng/μl)

Test: 21-08-2007

In vitro testing at 37°C

Fig.1: GFP Expression of pTet-GFP in vitro

The pTet-GFP contruct was tested in vitro using commercial S30 Cell Extract at 37 °C. As shown in Fig.1, pTet showed a marked increase in the first hour before levelling, reaching a slow but steady increase throughout a period of 4 hours.

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

pTet-GFP in vitro GFP expression at 37°C

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

pTet-GFP in vitro expression at 10°C

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. The positive control shows a decay over the four hours, however because of the short sampling time we cannot tell whether this decay is expotential. 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

pTet-GFP in vitro GFP expression at 45°C

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 shows decay, we cannot tell if this decay is expotential because of the short sampling time. 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)

Test: 21-08-2007 to 22-08-2007

37 Degrees

21-08-2007- Results of pT7 at 37°C

The pT7 was tested in vitro in commercial S30 Cell extract at 37oC for 4 hours. The samples initially showed a higher fluorescence than that of the control, however, both control and samples decreased. The positive control showed a decay over 4 hours of measuring. The graph on the left 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).

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

The plate containing the samples was stored in a 37oC 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. The graph legend is the same as the one of the graph above. The fluorexcence of the sample did increase over the 2 days, however, so did teh fluorescence of the negative control which leads us to think that thsi increase in fluorescence is due to the methodology. The positive control shows an decrease in fluorescence due to teh decay of GFP.

Complete set of results and raw data