IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 1.2

= In vivo Testing of pTet-LuxR-pLux-GFP Construct=

Aims
To determine if the pTet-LuxR-pLux-GFP construct works in vivo after induction of 1mM AHL concentration Tested on  17-08-2007

Materials and Methods
Refer to protocols page.

Test: 17-08-2007
Controls:
 * Positive control - diluted GFP solution of equal volume
 * Negative control - LB media of equal volume

pTet-LuxR-pLux-GFP
Fig.1 showed that the pTet-LuxR-pLux-GFP construct gave a good amount of expression of GFP (~83000), indicating that the construct is functioning well in vivo. The drawback of this construct, however, is that LuxR levels within the cells are inconsistent, to which this variation would complicate the kinetics of GFP expression. Thus it will be unfair to relate the increase in expression of fluorescence solely due to the increase in induction and strength of the pLux promoter.

There was also significant variability in the results across the different samples, which could be attributed either to experimental methodology, or the intrinsic nature of variability of expression in the in vivo chassis.

Conclusion
To conclude,
 * pTet-LuxR-pLux-GFP construct gave a strong fluorescent signal, indicating good expression of GFP in vivo.
 * LuxR levels are inconsistent throughout experiment.
 * Significant variability was found in all in vivo fluorometer experiments.