Part:BBa_K1141002:Experience

Here we have posted our experimental results regarding applications of KillerRed: its characterization and our choice of strains for expressing the protein.

Experimental Conditions

Choice of the E. coli strain

BW25113 (wild-type strain) bacteria transformed with BBa_K1141002 were shown to express the protein in response to IPTG induction. However, results of OD610 monitoring showed that these cells grew really slowly (r = 0.08 h^-1) as compared to untransformed WT cells (r = 0.77 h^-1). Repression of the pLac promoter by the endogeneous LacI repressor is not sufficient for preventing the expression of KR, a protein which affects cell growth even at low light levels.

The M15 commercial E. coli cell strain (Qiagen) in which the lacI repressor is expressed at high levels (due to the pREP4 plasmid) express the KR protein in response to IPTG and display a faster growth rate than the BW25113 cells transformed with BBa_K1141001 (Fig 4.). For this reason, M15 or other equivalent (high LacI expression) cells are more appropriate to experiment with BBa_K1141001 and BBa_K1141002.

Figure 4.
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.

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.

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.

Figure 5.
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

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 Fig 6.

Figure 6.
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.

This new experiment confirms that the expression level of 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.

Applications of BBa_K1141002

Bacterial Cell Killing:

On the graph above, an experiment with KillerRed using mCherry as negative control was performed. Cells expressing either KillerRed or mCherry with BBa_K1141001 (same sequence as BBa_K1141002) were inoculated into M9 minimal medium (recipe here). After a period of growth in darkness,

Bacterial Growth Regulation with light intensity:

The curves above show the evolution of culture OD610 and KillerRed fluorescence (relative light units) with BBa_K1141001 at different light intensities. The experiment shows that the cell killing and photobleaching effects are a function of light intensity: the higher the light intensity, the stronger the effects. Note that photobleaching (destruction of KillerRed's chromophore, making the protein colourless and non-toxic) is a phenomenon that is present in all fluorescent proteins but that KillerRed is particularly susceptible to photobleaching.

Finally, we can show that cells survive after illumination while keeping BBa_K1141001.

Usage of BBa_K1141002

Fluorescent Protein Characteristics:

KillerRed is a type of Red Fluorescent Protein, with the absorption and emission spectra shown above. Left peak is absorption, right peak is emission. The optical properties can be obtained from EVROGEN's detailed description. Credits to EVROGEN for the spectra.
It is advised to illuminate the protein with a large portion of the green spectrum, as illumination with green laser light at 535 nm has been reported to us as ineffective. In our experiments, we have illuminated it with a white LED light (INSERT REFERENCES HERE), and our observations showed the protein to be active when illuminated in this manner. Expression strain: During our experiments with KillerRed we used QIAGEN's M15 strain of E. coli. This strain is Thy- (no thymine production, meaning no vitamin B production). It needs to be grown in rich media or in minimal media with vitamin B as a supplement. Our experiments used M9 minimal media with added vitamin B (See Grenoble-EMSE-LSU 2013 wiki for exact media composition).
M15 cells contain the PLAC repressor plasmid pREP4 (Kanamycin resistance) which provides efficient repression of the notably leaky PLAC used in pQE30. This PLAC can be found in BBa_K1141001 and BBa_K1141002. M15 cells with the pREP4 plasmid and BBa_K1141001 or BBa_K1141002 do not express KillerRed until introduction of IPTG into the culture. The following experiment shows production of KillerRed as a function of IPTG concentration (millimolar):

Correct IPTG concentration

Using Bba_K1141002 with induction of PLac at saturating concentrations of IPTG (1 mM) has a severe effect on the gowth of cells, which is significantly slowed. During the 2013 Grenoble-EMSE-LSU team's project, we determined an optimal IPTG concentration to use for good KillerRed expression while avoiding any effect on the growth of cells, while the culture is kept in the dark.
Below is a comparison of growth curves obtained with BBa_K1141000 (mCherry negative control) and BBa_K1141001 (same sequence as for BBa_K1141002, but in the commercial pQE30 plasmid) at different IPTG concentrations:



Note the lack of effect on cell growth for all IPTG concentrations with BBa_K1141000 (PLac-RBS-mCherry). This is expected behaviour for proteins that are non toxic to E. coli. High IPTG concentrations with cells containing BBa_K1141001 will significantly slow their growth, which can be explained by KillerRed's intrinsic cytotoxicity, even in darkness. It can be obsrved that the best results obtained for cell growth and KillerRed expression with BBa_K1141001 were with a concentration of IPTG=0.05 mM. This was the optimal IPTG concentration we used in further experiments with BBa_K1141001.

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