What is Lss mOrange?
LSS mOrange, is a Large Stroke Shift protein that acts as a fluorophore. It absorbs light at 395nm and emits light at 509nm, as orange fluorescence. This protein is a genetic variant of DsRed, which come from a coral.
The protein from DsRed is known to have a long time of maturation.
Motivation for using Lss mOrange in the Bioreporter
We wanted to separate the excitation and emission wavelengths of the bioreporter to be able to distinguish better the emitted light from the LED light with our prototype 1. The better the separation between excitation and emission wavelengths, the easier we can find a non-expensive filter to remove the excitation light, and cleaner the signal.
eGFP, the fluorescent reporter we are using for the moment, excitation and emission wavelengths are too close to be distinguished correctly.
You can see our write up in an article on biodesign.cc: eGFP or not to eGFP.
LSS mOrange has a larger Stoke’s shift than eGFP:
|Protein||Excitation wavelength (nm)||Emission wavelength (nm)||∆λ (nm)|
Experiment for LSS mOrange characterisation
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 :
eGFP -> Half time of maturation at 37° : Not determined
LSS mOrange -> Half time of maturation at 37° : 2.5h
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.
EDIT after Pr. Van der Meer's feedback, about this project of experiment :
All the experiments we wanted to do allow us not to test LSS mOrange characteristics, but E.coli characteristics! The only one which can interest us in the case of LSS mOrange characterization is "How much time does LSS mOrange need to take its conformation compare to eGFP?". (see the chapter below).
Comparing LSS mOrange and eGFP
The goal of this experiment was to compare LSS mOrange and eGFP, the two fluorescent reporters produced by the GMO bacteria in presence of Arsenic.
We wanted especially verify that LSS mOrange needs more time to take its mature conformation than eGFP, according to this paper.
In this experiment, we used two different active strains of bacteria, one with eGFP and the other one with LSS mOrange as fluorescent protein.
We prepared some standard solutions with the following final concentrations of arsenic : 0, 4, 7, 10, 30, 50, 75 and 100 ug/L.
Then we mix together 100 uL of bacteria and 100uL of arsenic standard and we loaded them in a 96 wells plate.
After 2.5, 4 and 6 hour of incubation at 37°C in a rotary shaker, we measured the fluorescence and the absorbance of the plate using a fluorimeter.
We finally ploted the fluorescence/absobance in function of the arsenic concentration for eGFP and LSS mOrange.
We thus get the following graphs :
- After 2.5h of incubation :
- After 4h of incubation :
- After 6h of incubation :
With the previous graphs we see that LSS mOrange take more time to take its mature conformation than eGFP. We must wait about 6 hours of incubation to have workable results.
But the results are similar with the two bioreporters.
In conclusion, we can use LSS mOrange as fluorescent protein to detect arsenic as well as eGFP, but we have to let it more time of incubation to become mature.
DIY detector with appropriate wavelengths and filters have to be built for testing.