IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.1: Difference between revisions
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====Results update: 17/8/2007==== | ====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. The temperature used throughout the experiments was 25oC. | 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). | ||
[[Image:IC2007 Experiments Phase1 Protocol1-1Res1.PNG |thumb|left|500px| Results of in vivo testing of pT7 over a 4 hour period ]] | |||
<br><br> | |||
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. This allows us to conclude that the pT7-GFP contruct does not work in vitro.Two reasons were hypothesized: | |||
1). The bacteria cells were in stationary phase <br> | |||
2). The pT7 construct is not working. | |||
===[http://parts.mit.edu/registry/index.php/Part:BBa_I719022 '''pCI-GFP''']=== | ===[http://parts.mit.edu/registry/index.php/Part:BBa_I719022 '''pCI-GFP''']=== |
Revision as of 13:51, 2 September 2007
Results summary
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.
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).
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. This allows us to conclude that the pT7-GFP contruct does not work in vitro.Two reasons were hypothesized:
1). The bacteria cells were in stationary phase
2). The pT7 construct is not working.
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 . |