Difference between revisions of "Part:BBa K1141002:Experience"

Revision as of 20:40, 19 October 2016

Here we have posted our experimental results regarding applications of KillerRed and its characterization. Note that experiments were performed with M15 cells transformed with BBa_K1141001 (same protein generator as BBa_K114002).

Experimental Conditions

Experimental setup

KR fluorescence can be used as an indicator of the level of expression of the protein in a cell culture. Optical density (OD610) provides real-time information on the biomass of the system. However, Od600 cannot be used to distinguish living and non-living cells. This means that counting colonies on agar plates is a better method to quantify live cells when using BBa_K1141001 and BBa_K1141002.

Kinetics experiments were performed using 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: 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).

Figure 1.
Overview on the experimental set up used for KillerRed characterization.

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

We observed that when inducing KillerRed production with saturating levels of IPTG (1 mM), cells grew very slowly, meaning that expression of the protein affected our system in a significant way (see choice of the expression strain, top). In order to bypass this effect we made an experiment which would allow us to determine an optimal concentration of IPTG, to obtain good expression of KillerRed while avoiding any effect on cell growth.

The experiment was designed to induce KR expression with different concentrations of IPTG in a series of cultures, 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 Fig 2.

mCherry vs KR

Figure 2.
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.

We observe that KR expression increases with IPTG concentration. This new experiment also confirms that at high expression rates KillerRed has an effect on cell growth that isn't observed for a control red fluorescent protein, mCherry. This effect is threshold-based, meaning that if we go over a certain concentration of IPTG and thus a certain protein expression level, then the cells start growing much more slowly. We observe that at an IPTG concentration of 0.05 mM, there is no effect on cell growth compared to control, while protein expression at that concentration is the best out of all the curves. This experiment allows us to define the IPTG concentration used thereafter in KillerRed characterization: 0.05 mM.

Experimental results for BBa_K1141002

Response to a Constant Illumination (Proof that KillerRed works)

This experiment determines whether or not KR-expressing bacterial cells can be killed under illumination with white light, at constant intensity.

Our proof of concept experiment was performed with cells from an ON preculture resuspended in two different Erlenmeyer flasks, filled with 25 mL M9 medium, supplemented with 200 µg/µL ampicillin, 50 µg/µL kanamycin and 0.05 mM IPTG. The two cell samples were further incubated at 37°C, 200 rpm, while monitoring OD610 and fluorescence at 610 nm. One cell sample was illuminated at maximal intensity (P = 0.03 µW/cm²) from time point 180 min until the end of the kinetics experiment (740 min) whereas the second one was kept in the dark. Cells were plated on agar plates at each time point, using serial dilutions. Results of the cell plating are shown in Fig. 3.

Curves showing the decreasing number of viable KillerRed-expressing cells as a function of time when the culture is illuminated. A control culture kept in the dark keeps its number of viable cells

Fig. 3. Living cell density (cells/µL) as a function of time for both the dark (blue) and illuminated (red) samples. The growth rate for the illuminated sample starts decreasing at time point 240 min, 1 h after the light source is switched on. This corresponds to a decrease in the concentration of living cells.

Cellular Death is ROS-mediated

An additional kinetics experiment, using mCherry-expressing bacteria as a negative control, yields proof that KillerRed ROS production is the cause of bacterial death:

mCherry and KillerRed-expressing M15 bacteria were inoculated at OD610 = 0.015 in LB medium, supplemented with antibiotics and 0.05 mM IPTG. Cell samples were subsequently incubated at 37°C, 200 rpm. Fluorescence (540/630 nm) and OD610 measurements were performed every 20-100 min for 535 min. Erlenmeyers were kept in the dark for the first 180 min, and were then illuminated (P = 0.03 µW/cm²) for the rest of the experiment.

mCherry vs KR

Figure 4.
OD610 (A) and fluorescence (B) as a function of time for mCherry or 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 the dark, with growth rates of 1.39h^-1 and 0.57 h^-1 in early (0-120 min) and late (120-180 min) exponential phase, respectively. At t = 255 min a strong decrease in the growth rate of KR-expressing cells occurs as compared to mCherry-expressing cells. This phenomenon 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 [7]. Free radicals such as H²O² are highly reactive, and cause damage to endogenous proteins and DNA, ultimately leading to cell death. E. coli defense mechanisms against oxidative stress, including the superoxide dismutase and catalase enzymes [8], seem insufficient in preventing significant and irreversible ROS-mediated damages inside bacteria.

Cell Growth Recovery after Illumination

The following kinetics experiment shows the ability of cells to recover after a period of illumination corresponding to a period of oxidative stress. Light was applied for 120 min (P = 0.03 µW/cm²). 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 IPTG induction. The results are shown below on figure 5.

results

Figure 5.
OD610 (A) and Fluorescence (B) responses of a culture exposed to a 120 min constant light illumination (P = 0.03 µW/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 stimulation. This phenomenon occurs right after the beginning of the illumination (t = 210 min), the moment at which ROS start being produced and accumulate inside bacteria (Fig 5.B). Fluorescence of the illuminated cell sample still increases during illumination, probably because KR is still being produced by E. coli. There is thus a progressive accumulation of intracellular damages caused by oxidative stress during light illumination. A duration of 120 min of illumination seems long enough for the cell population to accumulate sufficient ROS damage. Indeed, at this illumination threshold, a significant decrease in the amount of living cells is measurable, ultimately leading to the stabilization of the OD610 between 365 and 510 min (Fig 5.A). The cells that have survived the light-induced oxidative stress divide again after time point 510 min.

Influence of Light Intensity

In these experiments we simply put an additional light source inside the incubator in order to illuminate two cultures at once, at 100% and 50% light intensity respectively. The light sources were switched on 195 minutes after inoculation, until the end of the kinetic experiment (600 min). Another sample of KR-expressing M15 bacteria was kept in the dark, as a negative control. Results of OD610 and fluorescence measurements are shown in Fig 6.

Figure 6.
OD610 (A) and fluorescence (630 nm) (B) as a function of time for 3 different bacterial cell samples, under different light conditions. The sample kept in the dark is represented in blue, the ones illuminated at 50 and 100% of the maximal intensity (Imax) in red and green, respectively. The light sources were switched on 195 min after inoculation, until the end of the experiment. Illuminated samples displayed similar fluorescence/OD610 ratios at time point 240 min (4945+/-49 RFU and 4465+/-182 RFU for 0.5*Imax and Imax, respectively). Error bars represent standard errors of duplicates.

Optical density values of the 3 bacterial cell samples start diverging 105 min after the light sources are switched on. ROS-mediated intracellular damages start accumulating inside the bacteria after t = 195 min, leading to a significant change in the number of living cells after t = 300 min (Fig 6.A). For all cultures, OD610 increases after time point 300 min, but at different rates that depend on the intensity of illumination.

According to our expectations, the sample in which the biomass increases the most is the one that was kept in the dark, the condition in which no ROS is produced by KR.
When illuminating the culture at half of the maximal intensity value, OD610 increases more slowly. At this light level, ROS production is likely to induce the killing of some of the bacteria of the population, and to slow down the division of the remaining viable cells. This hypothesis is confirmed by the fact that fluorescence never stops increasing during illumination (Fig 6.B), meaning that some of the cells are still alive and able to produce the KR protein.
At maximal intensity, the increase of the biomass is slowest and tends to stabilize after time point 540 min. In that situation, most of the cells are being killed. Since OD610 represents both living and dead cells and dead cells are not able to divide, a plateau is progressively reached. At time point 420 in, the fluorescence curve tends to flatten (Fig 6.B), a phenomenon that reinforces the hypothesis of the killing of a higher number of cells at maximal intensity than at lower light doses. Since the number of living bacteria decreases, less KR is produced, and the rate at which fluorescence increases begins to be limited due to photobleaching.

We conclude that KR-mediated phototoxic effects are light intensity-dependent.

12 W/m² Light Intensity - Exeter 2016

We improved characterisation of KillerRed by exposing cultures expressing the protein to previously untested light intensity. We compared the phototoxicity of KillerRed to the commonly used Red fluorescence protein (RFP). Once we had established the efficiency the kill swtich, ministat chambers were inoculated with samples of E.coli BL21 (DE3) with the plasmid coding for the protein to determine the robustness of the kill switches over time.

The samples were tested for phototoxicity by exposing them to 12 W/m2 white light from a 4x8 LED array for 6 hrs. Samples were then spread plated and colony forming units (CFUs) were counted. The part was carried on the pSB1C3 plasmid and transformed into E. coli BL21 (DE3). Samples that were induced were done so with Isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.2 nM.

We constructed a box around the LED array to prevent ambient light entering, and attached acetate colour filters to provide the desired excitation frequency. Access to the inside of the box was gained through an opening cut in the front. With help from Ryan Edginton, we used a portable spectrometer (Ocean Optics USB2000+VIS-NIR-ES, connected to a CC3 cosine corrector with a 3.9 mm collection diameter attached to a 0.55 mm diameter optical fibre) to measure light spectra and absolute intensity in the visible range.

The graphs below show the average percentage of viable cells for induced and uninduced samples after 6 hrs of exposure to 12 W/m2 of white light. CFU count for the control condition was treated as 100 % and viable cells calculated as a proportion of that value. CFUs were not counted above 300, any lawns were assigned the value of 300. Error bars represent the standard error of the mean. The average temperature in the light box was 38.63 °C.

Percentage viable cells after 6 hrs in the light box. BL21 (DE3) transformed with KillerRed (BBa_K1914003) is compared to a control with no plasmid. 10 ml falcon tubes containing 4.5 ml of sample were placed label down in the light box to allow maximum exposure to the light.

Percentage viable cells after 6 hrs in the light box. BL21 (DE3) transformed with KillerRed (BBa_K1914003) is compared to a control with no plasmid. 10 ml falcon tubes containing 4.5 ml of sample were covered in tin foil before being placed in the light box.

Continuous Culture fo KillerRed Kill Switch

We further characterised this kill switch by growing the culture in a ministat and carrying out the same testing procedure, illuminating induced cultures 24 hours after induction with100μM of 0.1M IPTG in the light box for 6 hours. CFU’s were counted to determine if the kill switch was successful in cultures grown in the ministat for 120 and 168 hours to test how long the kill switch remains functional.

Comparison of CFUs formed by KillerRed exposed to light and kept in the dark for each sample taken from the ministat. The efficiency of the kill switch decreases over time as shown by the increasing number of CFUs.

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                                          <h3>Continuous Culture fo KillerRed Kill Switch</h3>
+<a href="https://parts.igem.org/Part:BBa_K1914003">BBa_K1914003</a>
                                          <p>We further characterised this kill switch by growing the culture in a ministat and carrying out the same testing procedure, illuminating induced cultures 24 hours after induction with100μM of 0.1M IPTG in the light box for 6 hours. CFU’s were counted to determine if the kill switch was successful in cultures grown in the ministat for 120 and 168 hours to test how long the kill switch remains functional.</p>