IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.1

Results Summary

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pTet-GFP

The pTet - GFP construct has been successfully tested in vivo. The levels of fluorescence of the construct where comparable to the levels of our positive control (200x dilution of GFP) meaning that GFP was actually produced. Therefore pTet-GFP works in E.Coli.A 1 hour run (taking measurements every 2.5 min) was also conducted to test the variation on the expression levels of pTet-GFP over time.

Results of testing pTet and pT7 at 0.15 sec counting time for fluorometer detector

Results of testing pTet and pT7 at 0.15 sec counting time for fluorometer detector

pT7-GFP

Initial testing of the pT7 - GFP contruct in vivo failed because it was not properly induced. It will be re-tested on the 17/08/2007 after it has been induced with IPTG and left overnight to allow for a measurable GFP amount to be produced. IPTG is a T7 inducer for the E.Coli strain used (BL21).

Results update: 17/8/2007

After re-testing the pT7-GFP in vivo, no positive fluorescence reading was observed. The fluorescence levels were recorded over a range of 4 hours for 3 repeats and compared, as in the case of pTet, to a positive control of diluted GFP. The temperature used throughout the experiments was room temperature (thermometer reading at 25oC).

Results of in vivo testing of pT7 over a 4 hour period

On the graph on the right we see the averaged-out fluorescence of the 3 repeats of the pT7-GFP assembly, compared to the diluted GFP solution used as a positive control. The fluorescence activity of pT7-GFP is minimal and remains at a constant basal level. This allows us to conclude that the pT7-GFP contruct does not work as fast as pTet in vivo or it does not work at all. Two reasons were hypothesized:

1). The bacteria cells were in stationary phase
2). The pT7 construct is not working.

A second test will be performed to test whether the low readings were due to extremely slow activation of the pT7 promoter or the inefficiency of the construct.

Results update: 21/8/2007

A second in vivo test of the pT7-GFP contruct has been performed to test if the construct actually works. The pT7-GFP was induced with iPTG and left overnight at a temperature of ???? . Three repeats were carried out to verify our results. The fluorescence reading was taken at the start of the experiment (21/8/2007 10:00 AM) , 7 hours later (21/8/2007 05:00 PM) and 24 hours later (22/08/2007 10:00 AM).

Results of in vivo testing of pT7 over a 24 hour period

From the graph on the right, we can conclude that pT7-GFP is not functioning properly. The graph shows the averaged out value of fluorescence reading of the 3 samples of pT7-GFP used as reapeats. Minute amounts of fluorescence have been recorded but it is 5 times less than the results recorded with pTet-GFP. It is now safe to conclude that the pT7-GFP assembly is not working in vivo. Further tests will be carried out in vitro as planned in a hope that using that chassis we will get better results.

pCI-GFP

Testing of the assembly has been postponed due to delays in the making of the construct. Testing will be carried out as soon as the construct is ready.

Results update: 21/8/2007

After numerous attempts to grow the pCI-GFP construct, it is found to be unavailable in the respository sent by the MIT and hence cannnot be grown and implemented in our experiments. Testing of this construct has been abandoned.

Complete set of results and raw data .