IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.1: Difference between revisions
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In addition we want to identify any problems with our testing method | In addition we want to identify any problems with our testing method | ||
Both constructs were tested ''in vitro'' on *pTet and pT7 ''in vivo'' [[IGEM:IMPERIAL/2007/Notebook/2007-8-17 | Tested 17-08-2007]] | |||
====37 Degrees==== | ====37 Degrees==== | ||
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Revision as of 07:22, 14 September 2007
In Vitro Tetsing
Aim
The aims of this experiment is to test the following constructs in vitro To Determine if the following constructs work in vivo:
In addition we want to identify any problems with our testing method
Both constructs were tested in vitro on *pTet and pT7 in vivo Tested 17-08-2007
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 (37oC). The initial test was carried out over a period of 6 hours, during which measurements were taken every hour. The results are shown below:
The graph on the right dispays the following.
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:
The graph on the right dispays the following.
10 Degrees
After successfully testing it at 37 oC, we determined that pTet-GFP it works (even if not at max efficiency) at a relatively high temperature. The next test is to see if it operates on the relatively low temperature of 10 o. To achieve this low temperature we placed a water bath in a cold room (at 4o) and set the thermostat of the water bath to 10 oC. The fluorometer plate was then loaded with the samples which include 3 repeats of the pTet-GFP in S30 cell extract, 1 -ve (cell extract + Nuclease free water) and 1 +ve control (diluted GFP). This was placed in the water bath with the well covered in sticky tape to prevent water contamination.
Readings of the fluorescence were taken every half hour for a period of 4 hours in total.
The graph on the right dispays the following.
What we can safely deduce from the graph is that at 10 oC the pTet-Gfp assembly is not working. A nearly constant basal level of fluorescence is observed which coincides with the -ve control (cell extract with nuclease free water only). This does not however coincide with our specifications that require the construct to be functioning up to 4 o
45 Degrees
Moving to the other temperature extreme, we tested wether our construct works at 45 oC, a temperature refrigerated meat will be rarely exposed but still it would be good to know the operating range of pTet-GFP. Again, the sample plate was loaded with the same configuration as in the 10 oC, covered with sticky tape and finally placed in a 4 oC water bath.
Readings of the fluorescence were taken every half hour for a period of 4 hours in total.
The graph on the right dispays the following.
The graph obtained by plotting the results Vs time is nearly identical to the one obtained for 10 oC. Again the activity remains minimal and at a constant level but slighty higher than the -ve control indicating that some expression was obtained but it was very limited. This drives us to the conclusion that 45 oC is too high of a temperature for the construct to work. Comparing data from all the above experiments, we observe that the optimum operating temperature of this construct in vitro is somewhere between 10-45 o. The exact value of it we will find out in phase 2 of the experiments.
pT7-GFP In Vitro (100ng/μl)
37 Degrees
The pT7 was tested in vitro for a span of 4 hours at 37oC 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.
The graph on the right dispays the following.
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 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 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 |
