Team:USP UNIFESP-Brazil/Measurement
AlgAranha Team USP_UNIFESP-Brazil
Measurements Standardization on Chlamydomonas reinhardtii
Although fluorescent proteins are often useful reporters in synthetic biology microscopy and expression evaluation, measuring fluorescence of recombinant proteins in photosynthetic organisms is a major challenge. Thus, native pigments constitute an additional source of noise to fluorescence signals, hindering more precise measurements of cell response. We performed simultaneous recordings of (i) absorbance, (ii) fluorescence of mCherry and (iii) chlorophyll, all in different concentrations of algae cultures and mCherry.
Taking advantage of our vector secretion capability, we used the supernatant of the algae expressing mcherry as a source of mCherry fluorescent protein. A 96 well plate was prepared with constant mCherry concentration per well, but with different cell concentration to evaluate the missing signal due to cell interference. The heat map (Figure 1) below demonstrated the plate layout and mCherry fluorescence data.
Figure 1: Heat map of mCherry fluorescence measurement vs different cells concentration.
From the heat map we observed that indeed cell concentration interfere with our fluorescence measurements. The values were adjusted, trying to understand the relationship between cells density and signal decrease. We used absorbance measurements to do that. Discounting blank measurements (From cells suspension without mCherry and no cells blank) and correlating the values with the absorbance data we obtained the Figure 2.
Figure 2: Missing mCherry signal per absorbance measurement. Trendline (point line) and its formula displayed, as well as R2.
Now we got a way to rectify our mCherry fluorescence measurements, even though further characterization need to be made to a finer correction. Figure 3, compares the result of our screen measurement prior to correction and after.
Figure 3: Comparison of uncorrected (top figure) and corrected (bottom figure) mCherry fluorescence signal.
The overall pattern was the same in this result, except by the absolute value in our measurements, as it would be expected, by the Missing mCherry graph (Figure 2). Since cells seem to be interfering in a direct manner with mCherry fluorescence, more experiments need to be performed to reach to a better fluorescence correction. Further on, mCherry concentration vs Cells dilution must be performed to check if this pattern is constant in different mCherry signal concentrations. Another possibility is to check the correlation between chlorophyll and mCherry signal removal. This kind of measurement are important for future studies of regulatory elements in Chlamydomonas reinhardtii and a trustworthy method must be in place to allow precise data acquisition.
This kind of measurement are important for future studies of regulatory elements in Chlamydomonas reinhardtii and a trustworthy method must be in place to allow precise data acquisition.
Interlab measurements:
Figure 4: All Test Devices after overnight growth on both M9 (back) and LB (front) media. From left to right: Negative Control, Positive Control, TD1, TD2, TD3 and controls for media auto-fluorescence, without bacteria.
For the InterLab track we have not only studied our Test Devices by standard plate reader, cuvette-based and flow cytometry assays, but also tested them for better measuring conditions (focusing on the comparison between LB and M9 media). We have also developed alternative measuring methods ranging from DIY-based (digital camera and fluorimetric-based methods) to single cell analysis by fluorescence microscopy. Moreover, we have evaluated the promoter strength of all devices by Relative Promoter Units using DH5a strain of E. coli harbouring all devices. Thus, we have fulfilled both the InterLab study and the extra credit requirements by searching for optimized measurement protocols and generating new cheaper and accessible approaches for assessing promoter strength. You can check our InterLab results and discussionHERE
