Team:Carnegie Mellon/Project/Results

From 2013.igem.org

Killer Red





KillerRed phototoxicity

Our photobleaching experiments showed the phototoxicity of KillerRed in pSB1A3:

Figure 1: Phototoxicity of KillerRed and mRFP1 (BBa_E1010) on E. coli XL10 by irradiating with a HBO100 lamp (includes 375 nm LP filter) for 5 hours.



KillerRed's phototoxic effect on E. coli XL10 is shown in Figure 1.

Triplicate cell viabilities are divided by the normalized fluorescence values to account for bleaching effects and the fact that KillerRed has a lower expression level than mRFP1. RFP 114% ± 20% (viable cells)/(Normalized RFU)
KillerRed: 53% ± 17% (viable cells)/(Normalized RFU)



KillerRed photobleaching curve

Figure 1: Photobleaching curve of KillerRed with a HBO100 mercury-arc lamp



XL10 Ultracompetent cells were transformed with KillerRed (BBa_K1184000) 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 1. Fluorescence Data is shown in Table 2.





Table 1 Tecan Safire2 Parameters

Excitation (nm)585
Emission (nm)610
Excitation bandwidth (nm)10
Emission bandwidth (nm)10
Gain129
Number of reads10
Integration Time (microseconds)40

Table 2 Shows the fluorescence data over time during photobleaching.

Time (minutes)Fluorescence (RFU)
042598
2037616
4033749
6029059
8025680
10021985
12019442
14017031
16015738
18013741

To read our conclusions and future directions, visit our Abstract and Future Directions pages.