Degradation Curve: Fluroescence of GFPmut3b as a function of time

Aims

To construct a degradation curve of GFPmut3b in the in vitro chassis. This will verify whether the degradation of the system would be a limiting factor to the expression of the system.

Materials and methods

Link to the Protocol

Results

Figure 1.1 Fluorescence vs time

Figure 1.2 Semi-log plot of fluorescence vs time

Discussion

From Figure 1.1, it can be seen that there is a lot of difference between the degradation of GFPmut3b in the samples. Each sample has a different concentration of GFPmut3b. The fluorescence of GFPmut3b increases and decareses many times for each of the sample averages. This may be due to the experimental errors in the samples like contamination of the samples or air bublbles in them.

Figure 1.2 is a semi-log graph of log of fluorescence against time. This plot allows us to calculate the decay constants for the different GFPmut3b concentrations.

Conclusion

The degradation constant was calculated from the semi-log plot. The average gradients of all the semi-log plots was -0.0003501. This is equivalent to a decay-constant of 0.0003501. Using the equation ln2/k = t_1/2, the half-life of GFP was calculated to 1980 minutes, or approximately 33 hours. This agrees with the literature value of ~24 hours. So for our experimental timescale of 6 hours, we would only observe 12% of our GFP to be degraded. This supports our assumption that the degradation of GFP is very small within the timescale of our experiments.

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