KillerRed Characterization
Response to a Constant Illumination
Comparison with mCherry: Cellular Death is ROS-mediated
We demonstrated in the previous section that KillerRed-expressing bacteria could be killed upon white light illumination. However, exposure to white light and incubation outside of the normal temperature range were shown to affect bacterial growth [1]. Therefore, we decided to perform additional kinetics, using mCherry-expressing bacteria as a negative control. Results of these experiments demonstrated that KillerRed is responsible for cell death in response to white light stimulations.
Construction of BBa_K1141000 : pLac-RBS-mCherry
mCherry is a red fluorescent protein that displays the same excitation and emission spectra as KillerRed [2]. Furthermore, this protein was shown not to be cytotoxic upon white light illumination [3]. For these reasons, the pSB1C3::pLac-RBS-mCherry biobrick (BBa_K1141000) was designed and
built by our team.
Kinetics
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Figure1. OD610 A and fluorescence B over time of mCherry and KillerRed expressing M15 bacteria. Constant light illumination at maximum intensity was applied from 180 min to 535 min. Temperature was measured in each Erlenmeyer during illumination and was shown to stay constant and equal to 37°C. The error bars represent the standard errors of 2 independent measurements. |
Figure1. OD610 A and fluorescence B over time of mCherry and KillerRed expressing M15 bacteria. Constant light illumination at maximum intensity was applied from 180 min to 535 min. Temperature was measured in each Erlenmeyer during illumination and was shown to stay constant and equal to 37°C. The error bars represent the standard errors of 2 independent measurements.
Both cell strains display similar growth dynamics in absence of illumination, with growth rates of 1.39h-1 and 0.57 h-1 in early (0-120) and late (120-180) exponential phase, respectively. Fluorescence data show that the concentration in KillerRed during this period increases exponentially while mCherry is not expressed yet, possibly because of differences between origins of replication in pQE30 and pSB1C3 plasmid backbones (HELP ! pSB1C3 ORI 500-700 copies against 300-500 for pQE30 ORI. Can it really come from differences between both promoters?).
At t = 255 min occurs a strong decrease in the growth rate of KR-expressing cells as compared to mCherry-expressing cells. This phenomenon, described in the previous section, is due to the killing of bacteria in response to light stimulations. Since the viability of mCherry-expressing cells is not affected, we conclude that KR is responsible for the decrease in the number of living bacteria when illuminating the sample with white light. Cell death is coupled to a decrease in the amount of fluorescing KR proteins. This phenomenon, known as photobleaching, was shown to be a good indicator of the amount of ROS produced by KR upon light illumination [3]. Free radicals such as H2O2 are highly reactive, and cause damage of endogenous proteins and DNA strands, ultimately leading to cell death. E. coli defense mechanisms against oxidative stress, including the superoxide dismutase and catalase enzymes [4], seem insufficient for preventing significant and irreversible ROS-mediated damages inside bacteria.
Cell Growth Recovery after Stopping Illumination
We showed that we could either increase or decrease the amount of living cells within our sample, by modulating the amount of light reaching the culture. KR-expressing cells were shown to be able to divide in the dark whereas they were killed upon appropriate illumination. But can the amount of living cells re increase after stopping illuminating the culture with light? In which shape are the cells that survive oxidative stress?
To answer the first question, we decided to perform another kinetic, in which a square light function (120 min, P = X W/cm2) was applied to the system. In this experiment, cells were inoculated at OD610 = 0.02 in 25 mL LB medium, supplemented with antibiotics and 0.05 mM IPTG. The first measurement was performed 30 min after induction.
Results
Figure2.
OD610 A and Fluorescence B responses to the system to a 120 min constant light illumination (P = X W/cm2). The illuminated sample is represented in red, the dark sample in blue. Error bars represent the standard errors of duplicates.
As mentioned before, photobleaching of KR is a good indicator of the cytotoxicity induced by this protein upon light stimulations. This phenomenon occurs right after the beginning of the illumination (t = 210 min), moment at which ROS start being produced and accumulating inside bacteria (figure 2.B). Fluorescence of the illuminated cell sample still increases during illumination, possibly because of KR still being produced by E. coli. This could be explained by progressive accumulation of the intracellular damages caused by oxidative stress during light illumination. 120 min of illumination seems enough for these damages to reach a threshold value, above which a significant decrease in the amount of living cells occurs, ultimately leading to stabilization of OD610 from 365 to 510 min (figure 2.A.). During this time, in absence of light stimulations, the cells that have survived oxidative stress divides. After 510 min of experiment, the number of living cells becomes high enough to trigger a significant increase in the amount of 610 nm light that is absorbed by the sample.
Then, it seems possible to recover a growth phase that follows the same dynamic as the culture that was kept in the dark during the whole experiment (figure 1. A). This phenomenon was called “growth recovery”.
Influence of Light Intensity
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