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 and pLac-RBS-mCherry

The KillerRed gene that we obtained initially was in an eukaryotic plasmid. To express KR in E. coli and characterize its effects in response to light stimulations, we decided to clone KR into the commercial prokaryotic expression vector pQE30 (Qiagen). This plasmid contains a pLac promoter and a Shine-Dalgarno Ribosome Binding Site (RBS) that allow gene expression in response to the presence of Isopropyl β-D-1-thiogalactopyranoside (IPTG). The pLac-RBS-KR sequence in the pQE30 vector has been submitted as BBa_K1141001. Furthermore, the pLac-RBS-KR sequence was cloned into the pSB1C3 plasmid, to give the biobrick BBa_K1141002. Both BBa_K1141001 and BBa_K1141002 were sent to the standard registry part (Fig. 1).

The choice of an inducible promoter is linked to the absence of literature about the effects of KR on cells in low light. Since KR could be cytotoxic and prevent bacteria from growing even at low doses of light, we wanted to be able to control its intracellular concentration. A negative control for KR characterization was also required. We decided to use the fluorescent protein mCherry, which displays the same excitation and emission spectra as KillerRed [1], and was shown not to be cytotoxic upon light illumination [2]. pSB1C3::pLac-RBS-mCherry (BBa_K1141000) was thus constructed from the existing biobricks BBa_R0010 and BBa_J06702 (Fig. 1).

Figure 1.
Figure 1. Biobricks BBa_K1141002 A and BBa_K1141000 A used for characterizing KR. C. Picture of KR and mCherry-expressing bacteria.

Experimental Protocol for KillerRed Characterization

Choice of the E. coli strain

We first decided to characterize KR in BW25113 bacteria, a wild-type (WT) strain derived from E. coli K12. Cells were successfully transformed with pQE30::KR and were shown to express the protein in response to IPTG induction. However, results of OD610 monitoring showed that BW25113 cells transformed with pQE30::KR grew really slowly (r = 0.08 h-1) as compared to WT cells (r = 0.77 h-1). One hypothesis was that repression of the pLac promoter by the endogeneous LacI repressor was not sufficient for preventing the expression of KR, a protein that could have affected cell growth even at low light levels.

We thus decided to switch to M15 cells (Qiagen), a commercial strain in which the lacI repressor is expressed at high levels from plasmid pREP4. M15 cells did express the KR protein in response to IPTG addition and displayed a faster growth rate than the BW25113 cells transformed with pQE30::KR (Fig. 2). For this reason, M15 cells were chosen to characterize KR.

Figure 2.
Comparison between the growth of pQE30::KR-containing BW25113 and M15 cells (without IPTG and in the dark). Cells were pre cultured ON in LB medium, supplemented with antibiotics. They were further re suspended in M9 medium, supplemented with antibiotics at OD610 = 0.1. OD610 was subsequently monitored in a 96-well plate for 600 min, using the Tristar LB941 microplate reader (Tristar, Bad Wildbad, Germany) available in the lab. Error bars represent the standard errors of 4 independent measurements.

Choice of the Culture Conditions

Experimental setup

For characterizing the effects of KillerRed on E. coli viability in different light conditions, we decided to focus on 3 kinetic variables: KR fluorescence, OD610 and colony forming units.

First of all, KR fluorescence can be used as an indicator of the level of expression of the protein in our cell culture. Then, optical density (OD610) provides real-time information on the biomass of the system. However, it cannot be used to distinguish living and non-living cells. This is the reason why the number of colonies growing on agar plates was considered to be able to quantify live cells with an independent technique.

Since the spectrophotometer available in the lab was not suitable for illuminating cell samples for extended periods of time, we decided to perform kinetics in 100 mL Erlenmeyer flasks, incubated at 37°C, 200 rpm. A LED light source, interfaced to a computer via a microcontroller, was placed into the incubator for illuminating cell samples. A customized software enabled us to tightly modulate the intensity of the light emitted by the source.

During most of the kinetic experiments, 800 µL of medium were pipetted every 30-60 min. OD610 measurements were performed using a GENESYS 6 spectrophotometer (Thermo Scientific, Waltham, MA, USA) whereas fluorescence was measured with a Tristar LB941 microplate reader, equipped with a 540/630 nm filter set for excitation and emission. Bacterial cell plating on agar plates was also performed at each time point, using serial dilutions.

Growth medium

M9-glucose medium was privileged in our experiments. As a matter of fact, it displays very low auto fluorescence and contains a single carbon source - glucose – hence providing more repeatable results than Luria-Bertani (LB) medium. pRep4 and pQE30::KR are respectively kanamycin and ampicillin-resistant, and these antibiotics were used at 50 µg/µL and 200 µg/µL.

IPTG induction

One important point for our project was to reach a high level of KR expression, without slowing down cellular growth. As a matter of fact, to increase or decrease the amount of viable cells in our culture, we needed to make sure that the bacteria expressing KR could grow in the dark and be killed in response to light stimulations. Now, the more KR is present inside bacteria, the more ROS are produced upon illumination and the more likely the cells are to die. But is bacterial growth affected by high intracellular concentrations of KR? Is there an optimal IPTG concentration to reach high levels of KR without disturbing cell division?

To answer these questions, we decided to induce KR expression with different concentrations of IPTG, while monitoring OD610 and fluorescence. M15 cells transformed with pSB1C3::pLac-RBS-mCherry (BBa_K1141000) were used as a negative control. To evaluate the amount of KR proteins per living cell, we normalized fluorescence by optical density. Results are shown in figure 3.

Figure 3.
OD610 (A and C) and Fluorescence/OD610 ratios (B and D) as a function of time for KillerRed (left panels) and mCherry (right panels)-expressing E. coli. The curves obtained with different concentrations of IPTG are represented in different colors as indicated on the legends at the right.

CONCLUSION WITH O.O5mM IPTG ! ADRIEN, could you please conclude to be sure that the english is correct ? The idea is to say that among the concentrations in IPTG providing normal cellular growth, the one giving the highest Fluorescence/OD610 ratio (0.05mM) was chosen, to get as many intracellular KR proteins as possible. THANKS!!!

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

Figure 1.
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