Biology

Light-Controlled Cell Density

The KillerRed Protein

KillerRed is our project's star protein and the key to our bacterial cell density control system. It represents the light-sensitive element that allows the cells to receive signals from the control device.

Main Functions

KillerRed is a red fluorescent protein [1], meaning that by illuminating it with wavelengths from a certain portion of the visible spectrum, it re-emits light in another portion with longer (less energetic) wavelengths. Below is the absorption and emission spectra for the KillerRed protein:

The KillerRed protein absorption (left peak) and emission (right peak) spectra
Source:Detailed KillerRed description from Evrogen

From the emission and absorption spectra, we can determine that the protein absorbs in the green portion of the spectrum with a peak at 585 nm and emits in the red portion of the spectrum with a peak at 610 nm, hence the name "KillerRed".
Emitted light from bacteria is proportional to the amount of protein in the cells. This allows for measuring protein concentration in a cell culture.

The most interesting function of the protein however is that it emits ROS (Reactive Oxygen Species) when fluorescing.[1]
ROS are highly unstable and react chemically with many substrates including proteins, lipids and DNA. These reactions are oxidative and damage the affected molecules, making ROS toxic to the cell. With sufficient amounts of ROS, a cell's essential components can be damaged beyond repair, and the cell killed. Thus illuminating KillerRed-expressing cells with light in the green portion of the visible spectrum kills them, a mechanism that we use to control cell density in a culture.

Structure

In order to understand why KillerRed has its unique properties it is necessary to look at its structure. The protein is remarkably similar to other fluorescent proteins like GFP (Aequorea victoria> and dsRed (Discosoma striata), featuring a beta-barrel housing a central alpha helix with the fluorescent chromophore at its center[2]. Normally the chromophore is protected from the outside environment by the protein shell, but this isn't the case with KillerRed.

A comparison of the 3D structures of monomerix dsRed (left) and dimeric KillerRed (right)
Credits to Carpentier P., Violot S., Blanchoin L., Bourgeois D. for the KillerRed structure, and Strongin D.E., Bevis B., Khuong N., Downing M.E., Strack R.L., Sundaram K., Glick B.S., Keenan R.J. for the dsRed structure.
Source: RCSB protein database entries 2WIQ and 2VAD.

KillerRed is a 240 amino acid protein with a 3D structure similar to other fluorescent proteins, with an eleven-strand beta-barrel surrounding an alpha-helix containing the chromophore, source of the protein's fluorescence and photoxicity.
KillerRed has a DsRed-type chromophore formed with residues 67Q (glutamine), 68Y (tyrosine), and 69G (glycine), to make QYG. The corresponding coding sequence can be found at the code segment CAGTACGGC.

The interesting properties of the protein are directly related to a unique structural difference among fluorescent proteins, consisting in an open channel linking the chromophore to the environment outside the protein. According to litterature, this is the reason KillerRed is able to produce 1000-fold more reactive oxygen species compared to EGFP which is another ROS-producing fluorescent protein.[2]

Origin

KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.[1]

Construction of pLac-RBS-KR

This section of the wiki describes the initial construction of a prokaryotic genetic network allowing the expression of KillerRed in E. coli

The KillerRed gene was in a eukaryotic plasmid when we obtained it initially. To get expression of the protein in E. coli, it is necessary to transfer this gene into a prokaryotic plasmid. The choice of the plasmid and the genetic components inside (Promoter, RBS) is particularly important because we want to control how fast KillerRed kills the cells. To do this we must control the amount of protein in the cells. There is no literature about the effects of KillerRed on cells in low light, so we suspect KillerRed could be toxic even at low doses of light. This represents an additional factor we had to take into account during the construction of the plasmid.

Components

Since the protein could be toxic, we want to control when KillerRed is expressed in the cells, and so we have to choose inducible promoters. In this way, even if cell growth is slowed by KillerRed we can easily measure its effects by plating the culture on agar plates and then counting the number of colonies. But to do that with some precision, and to obtain enough protein for the phototoxic effectwe could make sure that we had a significant cell population in the culture before expressing something potentially toxic. We then had two different inducible promoters to choose from

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

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