I cloned a GFP protein in to three ribosome mutants in order to compare the rate of translation between these three mutants. I used a 96 well plate and a plate reader that measures fluorescence and OD 600. I took measurements every 15 minutes for 8 hours, than I calculated the ratio between Flu and OD. Now I would like to calculate the rate of translation. What is the right mathematical way?
Firstly, please note that GFP needs considerable time to mature. The time needed depends on the particular GFP variant you use. Therefore the fluorescence does not reflect the protein amount.
Secondly, 8h incubation is long, growth of your culture slows down and mRNA concentration is changing. Therefore it is impossible to calculate the rate of translation.
Hi Tanel and thank you for your answering.
Can you give me a suggestion for what I need to do in order to use the experiential system that I have in order to calculate the rate of translation?
The GFP that I am using is a superfolded GFP which its sequence is optimized to E. coli
What you need to do is following:
1. Prepare cell extracts (S30) from all your mutants and control.
2. Prepare mRNA of GFP (by transcription in vitro; the best is to use turboGFP if you have it).
3. Establish in vitro translation (up to saturation level).
4. Perform in vitro translation for all mutants up to 25% of saturation and introduce GFP maturation correction.
5. Compare obtained results.
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