Part:BBa_K3900021
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mCRISPRed - improvement of mRuby3 |
This part is an improved version of the red fluorescent protein mRuby3 (BBa_M50009), that displays an enhanced stability at low pH conditions. The original part can be found at: https://parts.igem.org/Part:BBa_M50009
Measurement
The improved pH stability was determined by measuring the relative fluorescence of mRuby3 and mCRISPRed at different pH conditions. The fluorescence proteins were expressed in Escherichia coli BL21 DE3 and purified via IMAC beforehand.
After purification, both protein eluates were rebuffered and concentrated in PBS. The samples were diluted to the same concentration, before fluorescence measurement at different pH conditions using the TECAN plate reader.
For direct comparison the results are displayed as relative fluorescence intensity with the respective fluorescence intensity maximum at pH 4.6 for mRuby3 and pH 3.6 for mCRISPRed. Throughout pH 6.5 to 3, the relative fluorescence intensity of mRuby 3 and mCRISPRed is similar. However, for conditions of pH 3 and lower the relative fluorescence intensity of mRuby3 drops rapidly from 87 % at pH 3.6 to 8 % at pH 3. For mCRISPRed the relative fluorescence intensity declines stepwise from pH 3.6 to 2, with the final detected value being at 33 %. At this pH level, no significant fluorescence intensity of mRUBY3 could be detected. Students t-test proved the differences in relative fluorescence to be highly significant for pH 3; 2.5 and 2 (p < 0.001). With these results we confirm an increased pH stability of mCRISPRed in an acidic environment compared to mRuby3.
| emission | 592 nm |
| excitation | 460 nm |