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Experiments for LSS mOrange characterization
The goal of this set of experiments was to characterize LSS mOrange (strain, sensitivity to arsenic, time for development, fluorescence activity…) to finally define if it is better to work with LSS mOrange or eGFP, the bioreporter actually used, as part of our prototype.
We also want to answer some questions :
- How does the relative fluorescence of LSS mOrange vary with arsenic concentration?
- Is the buffer pH ideal for the LSS mOrange and AsrR synthesis, but also for the bacterial growth?
- How much time does LSS mOrange need to take its conformation compared to eGFP?
To answer this question, we proposed some experiments. Because of a lack of time and schedule conflicts, we never tested it. These experiments are just a direction of research.
How does the relative fluorescence of LSS mOrange vary with arsenic concentration in function of time?
The goal is to get a very precise calibration curve of the relative fluorescence of LSS mOrange in function of arsenic concentration. As we noticed during our first set of experiments, fluorescence increases linearly when the arsenic concentration varies between 0 and 100µg/L and then it reaches a maximum. So we can make a calibration in this range.
A possible experiment : We have to perform an experiment with the following strains of bacteria : LSS mOrange (from the plate and from the stock), AsrR and with no bioreporter.
We can prepare samples with arsenic concentration varying between 0 and 100µg/L, every 10µg/L.
We will make a measurement of the fluorescence and the absorbance (OD600) with the fluorimeter every hour to make a precise curve of the relative fluorescence as a function of arsenic concentration.
Is the buffer pH ideal for the LSS mOrange and AsrR synthesis, but also for the bacterial growth?
The goal of this experiment is to find the optimal solution which allows LSS mOrange and AsrR, the receptor of arsenic at the surface of E.coli, to get their active conformation and also allows an ideal growth environment for the bacteria.
A possible experiment : We have to perform an experiment with the following strains of bacteria LSS mOrange (from the stock and from the plate), AsrR and with no reporter.
We can prepare growth samples at different pH, by adding acid or basis, for example one solution every 0.5, between a pH of 2 and 10. We will make a measurement of the fluorescence and the absorbance with the fluorimeter every hour.
How much time does LSS mOrange need to take its conformation compare to eGFP?
According to the paper “A guide to choosing fluorescent proteins” (Shaner, N. C., Steinbach, P. A., & Tsien, R. Y. (2005). A guide to choosing fluorescent proteins. Nature methods, 2(12), 905-909.), we want to verify the following result for LSS mOrange and find a value for eGFP :
|
Protein |
Half time of maturation at 37° |
|
eGFP |
Not determined |
|
LSS mOrange |
2.5h |
Table : Half time of maturation of eGFP and LSS mOrange
A possible experiment : We have to perform an experiment by comparing the two strains bacteria LSS mOrange and eGFP. We will make a measurement of the fluorescence and the absorbance with the fluorimeter every 30 minutes between 0 and 7 hours, to see the evolution of the relative fluorescence as a function of the time of incubation.
For this experiment we got a feedback from Dr. Van der Meer. He asked us if it is very interesting in the context of our project to have a protein which becomes very quickly folded and active. In fact, between the time we collect some water and the test time, we can conserve the water sample by adding to it some basis (without loss of properties) and we have the time to make some manipulations with the bioreporter. So time of maturation doesn't seem to be essential. LSS mOrange normally has a longer time of maturation than eGFP, but it is finally not an argument to choose eGFP over LSS mOrange.