Fluorescent proteins are important markers to observe processes in vivo and in vitro. However, due to a lack of standardization, measurements using these proteins are often hard to replicate and comparing data universally is almost impossible. Within iGEM, the measurement committee worked together with many teams to establish a standard for GFP measurements: using fluorescein as a method of standardization. While this is great for many applications, using GFP might not be the best or only choice for all projects. Since we believe that standardization needs to be introduced for more fluorescent proteins, we extended the use of the Relative Fluorescence Unit beyond GFP. Working together with the measurement committee and several other teams, we established Texas Red as a reference dye for mCherry measurements in E. coli and S. cerevisiae, enabling all teams working with red fluorescent proteins to generate reliable and reproducible data.
The Fluorescent Protein mCherry
All mCherry parts in the iGEM registry combined is has been used 494 times. While GFP had been used approximately 350 times more, the relevance of mCherry is on the rise. Just this year four teams contributed to better characterize mCherry, resulting in better results and experimental designs due to more prior knowledge. Moreover, it has been easier and more accurate to use GFP due the previously conducted standardizations. As mCherry is being used particularly often in the iGEM community, it is highly relevant to develop a standardization technique for mCherry-measurements. The high popularity of mCherry originates from the fact that it is bright enough to provide a strong signal which is reliably and easily detected and imaged. Moreover, mCherry does not have any toxic effects on cells (Ali, Ramadurai, Barry, & Nasheuer, 2018), enabling live-cell-imaging (Ettinger & Wittmann, 2014) and the relative comparison of promoter strengths (Schikora-Tamarit et al., 2018). Additionally, it is possible to easily detect a change in protein concentration, if the respective proteins are tagged with mCherry (Duellman, Burnett, & Yang, 2015). This is also simplified by its monomeric structure, allowing for a clear signal without impacting the general structure of the protein of interest (Shaner et al., 2005). These described factors allow for mCherry to be a good reporter protein for many applications, therefore explaining its broad usage. However, a common problem is the comparison of several studies using mCherry since the measurements are always done relatively, rather than absolutely. While this was – and still is – a problem for many fluorescent proteins, a solution has been found for GFP. Over the last few years, the iGEM measurement committee and many iGEM teams worked together to establish fluorescein as a standard for GFP measurements, enabling scientists all around the world to convert their relative data to absolute numbers with units and facilitating collaborations among labs. Even though it is great to have units for one fluorescent protein, using GFP might not be the right or only choice for many projects. In some cases, more than one fluorescent protein might be needed to enable the realization of a project. We were met by this challenge when trying to compare the strength of two promoters within one cell. Therefore, we started looking into alternatives for GFP and decided to use mCherry.
A red flourophore: Texas Red
To improve the utilzation of mCherry measurement standardization needed to be done. After looking into several possible dyes as a comparison for mCherry, Texas Red (or sulforhodamine 101 acid chloride) came to our mind. After comparing the absorbance and emission maxima of several red fluorophores. Alternatives we considered were Rhodamine, Alexa or Rox (“Fluorophore selection—DE”). However, the absorbance spectra of Texas Red and mCherry were the most similar.
Determining Purification Efficiency
Determining protein stability
The photostability of mCherry
In order to gain better data on the effects of light exposure to mCherry we placed it in an environment with a medium light intensity, drawing samples from it repeatedly and measuring its fluorescence (figure 8). The stability of mCherry is rather high in the first minutes of light exposure. Even though the fluorescence is restricted to approximately 90% after 5 minutes, it does not decrease much further within the following 25 minutes of light exposure. Within the next 30 minutes, the intensity had decreased to approximately 60% of the initial intensity and after 2 h it was lower than 40% of the initial value. When the final sample was drawn after 3h of daylight exposure, the intensity was reduced to not even 20% of the initial value. After all, one can say that even though mCherry is sensitive to light and is affected by photobleaching, using it in a regular lab environment is still feasible as long as it is kept in the dark as long and often as possible. Since exposing it to medium levels of light decreases its fluorescence constantly, however only strongly noticeable after 40-60 minutes, one can assume that exposing it to light for a few minutes during purification and sample preparation for measurements does not compromise the reliability of the data recorded.
The pH-stability of mCherryNext to the photostability, we also investigated the pH-stability of mCherry by mixing it with buffers at several pH values and recording its fluorescence (figure 9). Detectable fluorescence can be measured in a pH-range from pH 4 to pH 12 while the pH-optimum seems to be 6-7. Interestingly, the fluorescence intensity seems to have a second optimum at pH 10-11. This can be explained by the disintegration of the protein, as we showed on our Contribution-page .
Observing endocytosisTo observe the endocytosis of certain proteins for the fungi we used as model organisms, we fused the proteins of interest to mCherry. A functional uptake of the fusion-protein would lead to a measurable decreasing concentration of the fluorescent protein in the medium. We thought it would also help us to predict, whether fusing the proteins to another particle would disable the cells from performing endocytosis. To test this for S. cerevisiae, we fused mCherry to Mating Factor α, Opy2 and Flo11, using a glycine-linker. After mixing the fusion-proteins and S. cerevisiae cultures and cultivating them for one hour in the dark the fluorescence of the supernatant was measured in 15-minute intervals. The recorded data was normalized to samples taken from proteins mixed with the cultivation medium for the same amount of time and treated equally (figure 10). Detailed information about its integration into our project can be found here. Using Texas Red as a reference allowed us to effectively compare the results from several experiments conducted for showing the efficacy of the endocytosis process. For A. niger, we tested the endocytosis by using a proline transporter, using the same method as for S. cerevisiae by measuring the fluorescence of the supernatant. Since A. niger grows way slower than Saccharomyces cerevisiae using Texas Red as a reference for all measurements would have enabled us to measure the uptake into Aspergillus niger over a longer period. However, due to the short timespan we had left, we could not perform any long-lasting experiments on A. niger (figure 11).
In vivo measurements
In vivo fluorescence: E. coliTo prove that you could also use Texas Red when measuring fluorescence in cells we constructed five plasmids on which the expression of mCherry-His is regulated by promoters from the Anderson promoter library, namely:
Additionally, we regulated the expression of mCherry using the P8 promoter from the M13 phage (BBa_M13108) to further characterize it. To get a wider analysis of our measurement method we also invited other teams like iGEM Duesseldorf iGEM Duesseldorf and TU Darmstadt to characterize our mCherry parts.
In vivo fluorescence: S. cerevisiae
Mini Interlab Study
We also sent plasmids encoding for three different Promoter-mCherry-His combinations to the team Duesseldorf. Since we had already determined the promotor strength by comparing the fluorescence signal, a correlation of measurements by another team would be very interesting.
Figure 17 demonstrates the absolute absorbance units as they were recorded in the lab of team Duesseldorf and our lab. Even though both measurements were conducted in plate readers and the expression of mCherry was regulated by the same promoters, it seems like the fluorescence was way lower for the measurements conducted in Duesseldorf. Without normalization the differences in the fluorescens level is traced back to the different E. coli strain tested. That would mean that ER2566 is a way better producer of mCherry than DH5Α and BL21. However, as soon as the data is normalized to a certain concentration of Texas Red, representing a known variable in the system, the differences shrink. While there are still some differences between the fluorescence intensity of figure 17 and 18, they are minimized in comparison with figure 18. Therefore, one can assume, that the differences in fluorescence depicted in figure 18 are caused by biological differences, rather than measurement errors.
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