Part:BBa_K3900021

emission	592 nm
excitation	460 nm

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.

Figure 1:Relative fluorescence intensity of mRuby3 and mCRISPRed at different pH conditions measured in the TECAN plate reader, significance differences are marked with *** for high significance (p < 0.001)

Figure 3: Figure 3. DLS of the heat-purified fractions obtained from cell lysates of E. coli BL21(DE3) expressing T. maritima encapsulin monomers with the HexaHistidine tag. The bacterial cultures were incubated at a post-induction temperature of 37°C (A) and 18°C (B). The peaks revealed the presence of monomers (diameter ~ 1 nm) and aggregates (diameter > 100 nm) of the protein construct. The absence of a signal at 20-24 nm indicated that there were no assembled 60-mers encapsulin cages in the heat-purified fractions.

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.