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In vitro Testing of pT7-GFPmut3b Construct

Aims

To determine if the pT7-GFPmut3b(INSERT LINK) construct works in vitro. In addition we wish to characterize this constructs response at varying temperatures.

Materials and Methods

Link to Protocol

Results

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.

Controls:

• Negative Control-pT7 construct (LINK)

Constants:

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

Discussion

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 greater than 200 minutes.

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

Conclusion

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