Imperial/Wet Lab/Results/Res1.9


In vitro Testing of pT7-GFPmut3b Construct


To determine if the pT7-GFPmut3b construct works in vitro. In addition we wish to characterize this construct's response at varying temperatures.

Materials and Methods

Link to Protocol


Fig.1:pT7 in vitro - pT7 construct was tested in vitro for 360 minutes at 4°C, 25°C and 37°C. The fluorescence was measured and converted into molecules of GFPmtu3b in vitro using our calibration curve. The standard deviation is also shown for each temperature, which is an average of three samples. However, for 25°C one of the three samples was excluded because when compared to the other two samples it was clearly an anomaly.


  • Negative Control-pT7 construct


  • DNA Concentration - 4µg
  • Total Volume - 60µl

Raw Data


Figure 1. shows us that the pT7-GFPmut3b synthesizes over time for all the temperatures tested. When the three temperatures are compared it is clear that 4°C is the minimum temperature for expression and that 25°C is the maxiumum. Interestingly 37°C is not the maximum for pT7 in vitro, with 25°C producing almost double the molecules of GFPmut3b at 37°C.

All three temperatures appear to stop synthesis at a similar level. Although it is difficult with such limited sampling to extract the exact time the synthesis stops, we can approximate it to be greater than 200 minutes.

The variation within the samples was different for each temperature. 4°C and 25°C showed limited variation and 37°C showed the greatest variation.


To conclude the following approximations can be made:
Energy Levels

  • Synthesis stops >200 minutes

Total Output

  • 4°C - 1.2x1013 molecules of GFPmut3b produced
  • 25°C - 4x1013 molecules of GFPmut3b produced
  • 37°C - 8.3x1013 molecules of GFPmut3b produced

Optimum Temperature

  • 25°C was the optimum temperature that we found.