Summary
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 3D-structure of mChery as determined by Shu and Remington in 2006.
The red fluorescent protein mCherry was developed in 2004 (Shaner et al., 2004) and is derived from DsRed which was originally isolated from the corallimopharia Discosoma sp. (Campbell et al., 2002).
By looking at the Google Trends for mCherry and green fluorescent protein (GFP), the interest seems to be similarly high, although GFP is the most traditional and still commonly used fluorescent protein.
The relative number of searches for the terms "green fluorescent protein" and "mCherry" on Google. The values have been determined with Google Trends.
Even though, until mCherry was published in 2006 there were little to no searches for it, the interest in it has risen quite quickly and now it seems to be as high, if not higher than for GFP.
Moreover, it looks like the interest in GFP is lower now than it was before mCherry was discovered. Besides mCherry, the development of more new fluorescent proteins intensifies the lower interest in GFP. More chosable fluorescent proteins cause lesser importance of individual ones. However, as more and more new fluorescent proteins arise and the variety that is frequently used increases, the urge to develop methods for easy standardization of fluorescent measurements grows.
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
Emission- (dashed lines, marked with F for fluorescence) and excitation-spectra (solid lines, marked A for absorbance) of mCherry purified via IMPACT-Kit (dark purple) and His-tag (pink) were measured (λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to their maximum.
The absorption (red) and emission (blue) spectra of Texas Red were recorded with a Tecan plate reader. The absorption was measured from 350 nm to 800 nm and the emission from 600 nm to 850 nm.
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.
Its ideal excitation wavelength at 586 nm and emission of 615 nm (“Sulforhodamine 101 S7635,” n.d.) were rather close to the excitation wavelength of 587 nm and emission wavelength at 610 nm of mCherry (FPbase, mCherry). We also verified these values by measuring the absorption and emission spectra of mCherry as well as Texas Red with our plate reader several times (Figure 3 and 4).
Texas Red was established as a nontoxic dye in 1982 (Titus, Haugland, Sharrow, & Segal, 1982). It is often used for staining cells for Fluorescent-activated cell sorting (Rashidian et al., 2016), in fluorescence microscopy (Miller, Jarrett, Hassan, & Dunn, 2017) or immunohistochemistry (Kerry O’Banion & Olschowka, 1999). If it is dissolved in water, the sulfonyl chloride (SO2Cl) group is hydrolyzed to the negatively charged sulfonate (SO3-), forming sulforhodamine 101 (Goding, 1996). It does not affect the absorbance spectra, as its absorbance maximum is still at approximately 586 nm and its emission maximum at 615 nm (figure 4). This enables us and other possible users of Texas Red as a standard for mCherry measurements to dissolve it in PBS rather than other harder-to-handle or more expensive solvents.
The chemical structure of Texas Red
Standard curves recorded for fluorescein and Texas Red, using the Tecan reader infinite M200. They were recorded at the respective optimal emission and absorbance wavelenth (Texas Red: λEx=570 nm, λEm=610 nm; Fluorescein: λEx=494 nm, λEm=525 nm) Both were normalized to the highest concentration of the fluorophore.
For the first validation, we recorded standard series of Texas Red and compared it to the established dye fluorescein. Experiments show that both dyes are rather similar regarding the general course and the resolution over different concentrations up to 1 µM.
One usage of Texas Red was the characterization of purified mCherry fused to other proteins. These fusion-proteins were constructed to observe the endocytosis into the fungi that we employed as model organisms testing our Troygenics. To do so, we first had to characterize the fluorescence of mCherry on its own. To learn more about how we went about doing that, check out our Contributions page here .
Purified Proteins
Determining Purification Efficiency
Fluorescence intensity of the dilution series of the two mCherry variants.
Fluorescence intensity of a dilution series of mCherry purified via IMPACT-Kit (dark purple) and mCherryHis (pink) was measured (λEx=570 nm, λEm=610 nm) using the TECAN infinite M200 and normalized to the fluorescence intensity of 0.5 µM Texas Red at the same wavelength.
For our measurements, we purified mCherry as well as our fusion proteins from E. coli using two different methods: via His-tag and via IMPACT-purification. To compare the purification techniques, we did not only look into the final concentration reached by each method, but also compared the fluorescence per µM of protein. By using Texas Red as a standard for doing so, we were able to compare measurements that were not conducted at the same time. This allowed us to measure the fluorescence quickly after purifying them, without storing the proteins purified by one method to await the purification using the other. This normalization indicates a higher absolut fluorescence per µM of mCherry purified by a His-Tag then with the IMPACT method, although the total concentration was lower.
Determining protein stability
The photostability of mCherry
While mCherry is theoretically great for all kind of measurements, a general issue of fluorescent proteins is the general low photostability. However, the effects of regular light on mCherry in a regular lab setting, have not been analyzed enough to give a definite recommendation on how to use it, except from keeping it in the dark as long and often as possible.
Fluorescence intensity of mCherry in dependence on light exposure.
mCherry was exposed to normal daylight at room temperature. The remaining fluorescence intensity was measured at determinated time points (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the intensity at t=0.
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 mCherry
Next 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 .
Fluorescence intensity of mCherry in dependence on the pH.
mCherry was incubated for 5 minutes in several buffers with different pH. The remaining fluorescence intensity was measured (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red) using the TECAN infinite M200 and normalized to the intensity at pH 7.
Using Texas Red for the measurements for photo- and pH-stability allowed us to repeat them several times and to gather fluorescence values that were comparable among the replicas. It would also enable one to measure each sample immediately after drawing it, if no possibilities of storing them in a position with no light exposure or other factors that could influence the fluorescence are given.
Observing endocytosis
To 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).
Mat_mCherry, Opy_mCherry and mCherry are taken up by S. cerevisiae
S. cerevisiae was incubated in SD media (30 °C, 180 rpm, OD around 0.4, dark) over 1 h with 1 µM mCherry (grey), Mat_mCherry (dark red), Opy_mCherry (dark purple) and Flo_mCherry (purple). Every 15 minutes a sample was taken and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.
The fusion proteins are selectively taken up by the target A. niger.
A. niger was incubated in SD media (30 °C, 180 rpm, dark) over 1 h with 0.5 µM mCherry (grey), Mat_mCherry (dark red) and Pro_mCherry (blue). After 60 minutes a sample was taken and the remaining fluorescence in the supernatant was measured using the TECAN infinite M200 (λEx=570 nm, λEm=610 nm, gain calculated from 2.5 µM Texas Red). The results were normalized to the fluorescence intensity at t=0 and the fluorescence intensity of the negative control without cells.
However, constantly using Texas Red as a standard to compare the fluorescence against, allowed us to combine measurements conducted at several timepoints within our project into one big data set with real fluorescent values, relative to Texas Red.
Relative Promotor strength The Promotor strength of Bba_J23100, Bba_J23104, Bba_J23110, Bba_J23114 and the P8-Promotor from the M13-phage were determined within the E. coli strain ER2566. The strain was inoculated to an OD660 of 0.02 and cultivated for 6h. Samples were drawn at t=0 and t=6 and all were measured (yEx=570 nm, yEm=610 nm, gain calculated from 0.156 µM Texas Red) with the TECAN infinite M200.
The previously measured promoter strengths as noted in the Anderson Promoter collection. The values were taken from here . Since these values are not normalized against Texas Red, they are not depicted as RFU. The fluorescence is normalized to the strongest fluorescence during the establishment of the Anderson Promoter collection .
Apparently, in figure 12, BBa_J23104 was by far the strongest promoter, followed by Bba_J23100, the P8-promoter from M13, Bba_J23108, Bba_J23110 and Bba_J23114.
Comparing this to the results of the original Anderson library, it shows that the general trend is similar – except for BBa_J23104 which is supposedly a weaker promoter than BBa_J23100. This is not true according to the data we recorded in our experiments. Additionally, we learned that the P8-promoter is approximately as strong as BBa_J23100 and can therefore be considered a strong promoter for all applications.
In vivo fluorescence: S. cerevisiae
We also used Texas Red as a reference for fluorescence measurements of S. cerevisiae cells. The S. cerevisiae strain was transformed using the transformation vector pRS304-GalL-mCherry. Upon selection and verification of successful transformation, the GalL-promotor was induced using Galactose. As a negative control, we also observed the non-transformed strain.
The two strains were cultivated and their fluorescence was determined every other hour. For each measurement, Texas Red was also measured as a reference and all values were afterwards normalized to 0.5 µM Texas Red (figure 14).
Fluorescence of S. cerevisiae The fluorescence determined for S. cerevisiae carrying mCherry and the inducible GalL promoter. The data was measured (yEx=570 nm, yEm=610 nm, gain calculated from 2.5 µM Texas Red) with the TECAN infinite M200 and normalized to the fluorescence of 0.5 µM Texas Red at the same wavelength.
As demonstrated, the fluorescence is increasing over time in both S. cerevisiae strains. However, the surge in fluorescence was larger in the culture induced with Galactose. This suited our expectancies.
Mini Interlab Study
The interlab button
To further extend the use of Texas Red and gain knowledge on experiences of other people using it, we crafted a protocol for measurements involving Texas Red and mCherry. It can be found here . Prior to publishing it on our wiki, we conferred with the iGEM Measurement committee about important points we would have to consider. Upon reading our first draft protocol, they advised us to conduct all Optical Density measurements for organisms with red fluorescent proteins at a wavelength of 660 nm instead of 600 nm. This would be necessary, since mCherry and other red fluorescent proteins emit light at the approximate wavelength of 600 nm and could therefore interfere with the OD600 measurements. After improving this, we enabled all other teams to read and use our protocol freely. We also provided the iGEM-teams Duesseldorf and TU Darmstadt with Texas Red to use within their project. In exchange, they sent us their Texas Red standard curves, enabling us to compare the use of Texas Red among different teams.
Their standard series combined with ours can be found in figure 16. As shown in this figure, the Absorption Units recorded by the plate readers differ strongly. These results are an additional proof, that standardization is really necessary to compare the measurements of different laboratories. Data from the plate reader in Duesseldorf cannot be compared to the data collected in Darmstadt and Bielefeld. And while Bielefeld and Darmstadt look rather similar for this standard curve, that does not mean that it would be possible to compare all the data generated using these plate readers.
Texas Red Standard Curves These standard curves were recorded by the teams Bielefeld-CeBiTec, Duesseldorf and TU Darmstadt. The different Texas Red concentrations were prepared in at least triplicates and their fluorescence was recorded using different plate readers.
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.
The expression of mCherry regulated by different promoters measured by different teams: Not normalized The fluorescence of three different E. coli strains expressing mCherry regulated under three different promorters: BBa_J23104 and BBa_J23114 from the Anderson Promoter collection and BBa_M13108, the P8-Promoter of M13. Team Duesseldorf recorded the values for DH5α and BL21 and Bielefeld-CeBiTec ER2566.
The expression of mCherry regulated by different promoters measured by different teams: Normalized The fluorescence of three different E. coli strains expressing mCherry regulated under three different promorters: BBa_J23104 and BBa_J23114 from the Anderson Promoter collection and BBa_M13108, the P8-Promoter of M13. Team Duesseldorf recorded the values for DH5α and BL21 and Bielefeld-CeBiTec ER2566. Both teams calculated a standard curve for Texas Red fluorescence. The data given here is normalized for the fluorescence detected at 0.5 µM Texas Red.
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.
To conclude the mini interlab study it becomes obvious that standardization for mCherry-measurements is urgently needed to ensure that data is reproducible. By introducing Texas Red as one possible reference, we believe that the iGEM community will move one step closer to generating reproducible data.
