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.

Results

Test: 17-08-2007

Fig.1: Total Fluorescence of pTet-LuxR-pLux-GFP in vivo over 7 hours

Upon induction, the pTet-LuxR-pLux-GFP construct showed a significant fluorescent signal when compared to the negative control. In addition, the fluorescence levels were comparable to that of the positive control, indicating strong expression of GFP with the construct.

Controls:

• Positive control - diluted GFP solution of equal volume
• Negative control - LB media of equal volume

Discussion

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.