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In vitro Testing of pTet-GFPmut3b Construct for constant and step temperatures

Aims

To determine how the system (pTet-GFPmut3b) responds to a step increase or a step decrease in temperature, compared to a constant temperture, in vitro.

Materials and Methods

Link to the Protocols page

Results

Fig 1.1:Molecules of GFPmut3b synthesised on average over time for two samples at constant temperatures of 4&degC and 20°C and two with a temperature step (4-20°C and 20-4°C)

Controls:

• Negative control - empty vector (pTet promoter) with the cell extract

Constants:

• Temperature - 4°C and 20°C
• Total Volume - 60µl

Raw Data:

• Raw Data excel file

Discussion

The 'step' samples were moved from their original temperature to the other 180mins after the start of the experiment. As it can be seen from the results above, the temperature step has a very rapid affect on the fluorescence of the DNA construct.

Both samples at 4°C have similar number of molecules of GFPmut3b produced until 180mins. After the sample is moved to 20°C, the GFPmut3b molecules produced increases to a very high value.

Moving the sample at 20°C to 4° decreases the amount of fluorescent protein synthesised by a little amount. This is what was expected as the pTet-GFPmut3b construct showed linear relationship with temperature in the earlier experiments.

Conclusion

It can be concluded from the results that the pTet-GFPmut3b construct is sensitive to a step increase and step decrease in the temperature.