Team:Carnegie Mellon/mRFP1

From 2013.igem.org

Killer Red




mRFP1 characterization

The QYG-derived chromophore of the mRFP1 protein. (Z)-2-(2-(4-amino-1-imino-4-oxobutyl)-4-(4-hydroxybenzylidene)-5-oxo-4,5-dihydro-1H-imidazol-1-yl)acetic acid. The extended π-system is highlighted in red.

Characterization of the Photostability of mRFP1

Photobleaching curve of mRFP1 with a HBO100 mercury-arc lamp

XL10 Ultracompetent cells were transformed with BBa_E1010 cloned with BBa_B0034 as the RBS and BBa_R0010 as the wild-type lac promoter and induced overnight with IPTG.The overnight was bleached for 180 minutes with HBO100 (100W Mercury-arc lamp). Fluorescence data was taken using a Tecan Safire II with the parameters shown in Table 3. Fluorescence values are shown in Table 4.

Table 3 Tecan Safire II Parameters

Excitation (nm)585
Emission (nm)610
Excitation bandwidth (nm)10
Emission bandwidth (nm)10
Gain63
Number of reads10
Integration Time (microseconds)40

Table 4 Shows the fluorescence data over time during photobleaching.

Time (minutes)Fluorescence (RFU)
048694
2043083
4036842
6030239
8031281
10025273
12021467
14018081
16015251
18014427

References

Campbell,R.E., Tour,O., Palmer,A.E., Steinbach,P.A., Baird,G.S., Zacharias,D.A. and Tsien,R.Y. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99 (12), 7877-7882 (2002)