IGEM:IMPERIAL/2007/Experimental Design/Phase1/Results 2.2

pTet-LuxR-pLux-GFP

Testing at 25 ^oC

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Today we tested pLux in vitro using 3 different combinations of cell extract:

1. pLux with Commercial(CM) S30 cell extract and CM Premix
2. pLux with Homemade(HM) S30 cell extract and HM Premix
3. pLux with HM S30 cell extract and CM Premix

The idea behind the first combination is to see if pLux works in vitro since we already know our commercial extract is working with pTet. If pLux does work with the commercial extract but proves to be reduntant in the second combination, which utilises only homemade cell extract & premix, then we know that our homemade extract inefficient. Lastly, the third combination is to test whether our homemade cell extract will work better with the commercial premix rather than the homemade one.

The experiment was carried out over a period of nearly 6:30 hours, taking measuremets every half hour. Each combination had 3 repeats and 1 control. The average value of fluorescence for each combination is on the graph below:

Results of in vitro testing of pLux over a 6 hour period at 25^oC

The graph on the right dispays the following.

Fluorescence of the diluted GFP solution (+ve control).

The average fluorescence of the pLux samples(3) in CM cell extract + CM premix

The average fluorescence of the pLux samples(3) in HM cell extract + HM premix

The average fluorescence of the pLux samples(3) in HM cell extract + CM premix

The fluoresence of a solution containing only S30 cell extract and nuclease free water
(-ve control) .

From the graph 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.

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.

Experiment 1

• Concentration of pTet-LuxR-pLux-GFP = 100ng/ul