Part:BBa_K1141000:Experience

Our experiments on PLac-RBS-mCherry-double terminator concern control experiments for BBa_K1141001 (same as BBa_K1141002) in which we tested its production rate, eventual effect on cell growth, and photobleaching when exposed to a 6W 12V LED lamp (search for AMPOULE 6 Watts - COB HAUTE PUISSANCE - 520 LUMENS - MR16 (GU5.3) - 12v - BLANC NATUREL, the site is French). Since the promoter was PLac we expressed mCherry at different rates using different concentrations of IPTG (figure 1):

Figure 1:Curves for mCherry fluorescence as a function of time with different concentrations of the PLac inducer IPTG.

The experiment gives us data on the maturation speed of mCherry as well. As induction is made at t=0min and fluorescence starts appearing at around 100 minutes we can expect the chromophore maturation time to be around this time. Experiment to verify resistance of the protein against photobleaching (figure 2):

Figure 2:Curves for mCherry and KillerRed fluorescence with KR in darkness and under illumination as a function of time.

The experiment was done in 100 mL erlemeyers with cells containing either BBa_K1141000 (mCherry) or BBa_K1141001 (KillerRed) expressing plasmids. Both promoters are PLac and are induced with the same concentration of IPTG= 0.05 mM at time t=0. Cell strain is M15 and growth media is M9 minimal media. Shaking conditions are 37°C, 200 RPM in a closed incubator. The curves show that under illumination mCherry does not photobleach (the curve follows that of killerRed in the dark) whereas KillerRed is significantly photobleached, showing that mCherry is much more resistant to photobleaching than KillerRed.

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