IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.2
In vitro Testing of pTet-LuxR-pLux-GFP Construct
Aims
To determine if the following constructs work in vitro:
To determine the consistency and efficacy of using different combinations of cell extract:
- pLux with Commercial(CM) S30 cell extract and CM Premix
- pLux with Homemade(HM) S30 cell extract and HM Premix
- pLux with HM S30 cell extract and CM Premix
Since it has been ascertained that the commercial extract works with pTet, the pLux construct should work in the first combination. This is to be compared with that of the second combination to test for the efficiency of the HM extract. The third combination investigates the possibility of combining our homemade cell extract with the commercial premix rather than the homemade one.
Tested on 24-08-2007
Materials and Methods
Refer to protocols page.
Results
pTet-LuxR-pLux-GFP
Test: 24-08-2007
In vitro Testing of pTet-LuxR-pLux-GFP at 25°C
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From Fig.1, it seems that there is a very strong fluorescent signal with that of pLux construct,
we notice that pLux does work in vitro (with CM cell extract) and quite well since by the end of the 6.5 hours, its fluorescence has intersected with our positive control (diluted GFP). Unfortunately however, our homemade cell extract did not match the commercial one in performance despite both extracts having the same amounts of pLux DNA in them (20 ul). Our homemade commercial cell extract peaked at 5000 fluorescence units after just 1.5 hour from induction whereas the commercial one increased continuously for the 6.5 hours reaching a fluorescence of 15000 (x3 the efficiency). Finally, our homemade cell extract proved not to be compatible with the commercial premix since their combination resulted in the lowest performance, a basal level comparable with our negative control. |
Discussion
Conclusion
Concluding, pLux is shown to work in a vitro system although the commercial extract (and commercial premix) should be preferrred over our homemade extract to obtain maximum perfomance for this construct.
