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

In Vivo Tetsing

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Aim

To Determine if the following constructs work in vivo:

• pTet-GFP
• pT7-GFP

The testing was comprised of several tests:

• pTet and pT7 in vivo - Tested 15-08-2007
• Retesting pT7 in vivo - Tested 17-08-2007

Results

Test: 17-08-2007

17-08-2007- Results of pTet and pT7

17-08-200707- Results of pTet in vivo over 1 hour

The results show that pTet works in vivo whereas the pT7 does not show any dramatic increase in fluorescence. Both the samples were compared to a negative and positive control to determine if there was any significant fluorescence. The fluorescence of pTet over 1 hour is shown, the results do show an increase. In addition it can be seen that there is a massive variation

Test: 21-08-2007

21-08-2007- Results of retesting pT7 in vivo repeat

21-08-2007- Results of restesting pT7 in vivo over 1 hour

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 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 or powerful as pTet in vivo or it does not work at all.

Complete set of results and raw data

Discussion

Test: 17-08-2007

pTet-GFP
The pTet construct works in vivo, however there are massive amounts of variation which are likely to be due to the experimental methodology. The error could be due the intrinsic nature of variability of the in vivo chassis or due to variation with the protocol i.e. measurement errors.

pT7-GFP
Initial testing of the pT7 - GFP contruct in vivo failed. We think that this could be because it was not properly induced. We need to add IPTG to a suitable concentration(~1mM) and grow overnight.

Test: 21-08-2007

pT7-GFP
This allows us to conclude that the pT7-GFP contruct does not work as fast or powerful 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.

We do not think it is the first reason because this is in the retest the protocol we followed ensures that the E.coli are in the groth phase.

Conclusion

We investigated the possibility that it was due to the fact that the pT7 consrtuct was not working. After investigating into the construct we found out that it has a weak ribosome binding site, this could explain the weak signal for the construct. We are going to look into cloning out the promoter and building our own construct.