In vivo measurements

In vivo fluorescence: E. coli

To 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:
psB1C3-Bba_J23100-mCherryHis<
psB1C3-Bba_J23104-mCherryHis
psB1C3-Bba_J23108-mCherryHis
psB1C3-Bba_J23110-mCherryHis
psB1C3-Bba_J23114-mCherryHis
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.

Fig. 1. 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 (y_Ex=570 nm, y_Em=610 nm, gain calculated from 0.156 µM Texas Red) with the TECAN infinite M200.

Fig. 2. 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.

Mini Interlab Study

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.

Fig. 4. 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.

Fig. 5. 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.

Fig. 6. 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 6 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.

Part:BBa_K2926264

In vivo measurements

In vivo fluorescence: E. coli

Mini Interlab Study