Part:BBa_K150009:Experience

ColicinE1 Producer Controlled by 3OC6HSL Receiver Device

Killing efficiency on bacteria

To measure the killing efficiency and which amount of cells or colicins are needed to reach any killing activity a colicin activity test was carried out. Therefore bacteria containing the colicin part (BBa_K150009 in TOP10 or MG1655) and GFP producing cells (reference promoter, TOP10) were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours and the optical density of the two strains was adjusted. The colicin cells were added in different ratios to a constant amount of GFP producing cells. The total volume was kept constant by adding TB-media (without antibiotics). The colicin production was induced by several concentrations (0 M-100 nM) of N-Acyl-Homoserin-Lactone (AHL). The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every 30 minutes for about 12 hours. As a negative control the similar test was carried out with cells containing the same plasmid without the colicin gene on it. (back)
Figure 2 shows results of tests with a prey-killer ratio of 100:1. Due to the correlation of the GFP intensity and the optical density, GFP intensity was used as marker for the living prey cells. In all experiments using killer cells and high AHL concentrations in the medium, the prey cells were killed completely. In reference experiments (see Figure 2, bottom) using E. coli TOP 10 cells harboring a LuxR-receiver without colicin operon (comparable to part BBa_T9002 without GFP), the prey cells were able to grow for each AHL concentration. Consequently the lethal action of the killing part could be shown. (back)

Figure 2: Results of colicin E1 toxicity tests for different AHL concentrations with a prey-killer ratio of 100:1. Top left panel: GFP intensities of prey cells treated with killer cells over time. Top right panel: Optical densities of prey cells treated with killer cells plotted against time. Bottom left panel: GFP intensities of prey cells treated with reference cells versus time. Top right panel: Optical densities of prey cells treated with reference cells over time. The graphs proof the toxicity of colicin E1 as well as the functionality of the part BBa_K150009. Each killer cell is able to kill up to 100 prey cell if there is a sufficient amount of AHL present.

Additionally it can be seen that the killing efficiency is related to the AHL concentration in the medium. Figure 3 shows a dose-response curve of GFP intensity after 12 hours dependent on the AHL concentration. The P_LuxR promoter has to be activated with an AHL concentration above 500 pM. Thus enough colicin E1 is produced and released to kill the prey cells (see Figure 3). (back)

Figure 3: Dose-response curve of AHL concentration and killing efficiency of the killer strain for a prey-killer ratio of 100:1. The GFP intensities at t = 12 h are plotted against AHL concentrations (0 M – 1 nM). For AHL concentrations above 600 nM all preys were killed.

Regarding the prey survival, dependent on the prey-killer ratio, several effects can be observed (see Figure 4). Having a prey-killer ratio of 1:1, or even a higher killer fraction, all prey cells were already killed when no AHL is present. This effect is caused by the leakiness of the P_LuxR promoter. For prey-killer ratios between 5:1 and 100:1 the killing efficiency can be regulated by the AHL concentration. In experiments with prey-killer ratios higher than 100:1 the growth of the prey strain was not influenced. (back)

Figure 4: Toxicity of LuxR-colicin E1-receiver part for different prey-killer ratios. The graphs show GFP intensities of prey cells at t = 0 h and t = 12 h. For prey-killer ratios of 1:1 all prey cells were killed after 12 hours even if there was no AHL present. For prey-killer ratios between 5:1 (not shown) and 100:1 the killing efficiency depends on the AHL concentration in the media. If there is a greater predominace of prey cells (500:1) the growth of the prey population is not influenced.

Killer-prey system

To measure the functionality the killer-prey system were tested. Therefore bacteria containing the colicin E1 part (BBa_K150009 in TOP10 or MG1655) and prey cells containing an AHL-producing part (BBa_K150000) were inoculated in TB-media with appropriate antibiotics at 37 °C for 4 to 6 hours. The optical density of the two strains was adjusted. The colicin E1 producing cells were added in different ratios to a constant amount of prey cells. The total volume was kept constant by adding TB-media (without antibiotics). The colicin production was induced by several concentrations (0 M-100 nM) of N-Acyl-Homoserin-Lactone (AHL). The OD and GFP intensities were measured at 37 °C in the Tecan Microplate Reader every 30 minutes for about 12 hours. As a negative control the similar test was carried out with cells, containing the same plasmid without the colicin E1 gene on it. Figure 5 shows that the prey cells were killed for prey-killer ratios from 1:1 up to 25:1. Therefore it is proven that our system works as expected. (back)

Figure 5: Killer-prey system test. GFP intensity of prey strains was measured over 12 h at 37 °C. Prey cells were killed for prey-killer ratios from 1:1 up to 25:1 (blue, green, red, light blue) due to the colicin E1 production of the killer strain. This production was activated by the AHL secretion of the prey strain. In a control experiment prey cells were able to grow (violet line).

Lysis of killing strain

Besides the killing efficiency the time course of the killer strain lysis was analyzed. Therefore growth curves of killer cells (BBa_K150009 in E. coli TOP10 cells) induced with different AHL concentrations were measured (see Figure 6).

Figure 6: Lysis test with LuxR-colicin E1-receiver dependent on AHL concentrations. On the left panel the growth of a killer population induced with different concentrations of AHL is shown. On the right panel the data of control experiments are plotted. For AHL concentrations between 0 M and 1 nM no influence on the growth of the killer population is visible. For concentrations between 5 nM and 25 nM the population grew for one hour, afterwards a lysis effect can be observed.

During the first hour of the measurement no effect can be observed. Afterwards cell lysis dependent on AHL concentrations is visible. For AHL concentrations between 0 M and 1 nM only few cells lysed. Thus growth of the population is hardly influenced. Concentrations from 5 nM up to 25 nM show a strong effect on the amount of lysed killer cells. For 5 nM and 10 nM the growth curves flatten out. After reaching a maximum during the third and fourth hour the population size decreases and converges to a constant value. For higher AHL concentrations, e.g. 25 nM, a similar effect can be observed, but it is much stronger. The population shows only a weak growing tendency but then converges directly to a constant value. In addition to these growth measurements lysis of the killer cells was observed under the microscope. Figure 7 shows killer cells which were induced by AHL (left panel, t = 0 min). After 30 minutes one cell is lysed (right panel). (back)

Figure 7: Visualization of the lysis effect of the killer cells. The prey (GFP) and the killer (mCherry) strains are mixed on a 0.5 % agarosepad. Killer cells were induced with AHL before adding to the medium. The killer cell on the left picture (white arrow) is lysed after 30 minutes. This effect is caused by the AHL induction. Colicin production and, at the same time in lower amounts, the lysis protein production leads to lysis of the host cell and colicin release.

Killing efficiency on eucaryotic cancer cells

The toxicity of colicin E1 on eukaryotic cells was tested by using breast cancer cells (MCF 7). They were inoculated with killer cells (BBa_K150009 in TOP 10) and reference cells (BBa_150009-like, no colicin E1 operon) for several hours.

Therefore MCF-7 cells (breast cancer cell line) were plated in 24 well plates (150 thousand cells per well) with 10 % FCS (fetal calf serum) DMEM (Dulbecco's Modified Eagle Medium) and incubated overnight at 37 °C, 5 % CO2. E. coli TOP 10 strains containing LuxR-colicin E1-receiver (BBa_K150009) and strains containing LuxR-receiver (no colicin) were grown overnight in TB media with appropriate antibiotics at 37 °C to an OD of 1.0 and diluted in the morning to an OD of 0.1. Once bacterial cells reached an OD of 0.35 they were harvested and inoculated on the MCF-7 wt and on MCF-7 GFP producing cells. http://2008.igem.org/Team:Heidelberg/Parts/Characterization back

Once MCF-7 cells were confluent (12 h after passage), E. coli TOP 10 bacteria were spinned down (1 ml of OD = 0.35) and resuspended with DMEM medium from the original wells containing the MCF-7 wt/MCF-7 GFP cells. A 24-well plate was used containing MCF-7 cells stained with Propidium Iodide (PI) alone, with LuxR-receiver cells not containing colicin and LuxR-receiver-colicin E1 cells. AHL was added to every well to a final concentration of 5 nM. Well content was collected at different time-points (0 – 6 h) and when necessary PI was added to the suspension 15 minutes before Flow Cytometer analysis.

At the referred timepoints, supernatants were collected and spinned down. Eukaryotic cells were detached by trypsinization (200 μl) for 10 minutes at 37 °C and then resuspended in 800 μl of KH + 1 % BSA (Krebs Henseleit medium with 1 % Bovine Serum Albumin). After resuspending, well content was added to the precipitated supernatant to account for floating dead cells. PI was added to each sample (1:1000) and incubated at RT for 15 minutes before acquisition. PI positive cells and GFP expressing cells (%) were measured for 3000 cells for each condition. Histograms and fluorescent measurements were obtained using a Cytomics FC 500 MPL from Beckman&Coulter.

MCF-7 cell death, measured by Flow Cytometry, was evaluated by % of stained cells with PI over the time, using colicin producing E. coli or strains which do not produce colicins as a control (Figure 1). E. coli was also quantified (data not shown). The PI measurements (filter FL3-620nm) were gated only to Mcf-7 cell population.

Figure 8 Positive PI MCF-7 cells (%) over time. E coli bacteria producing colicin E1 protein cause cell death at the earliest time point (2 h) and keep it at higher levels compared to the other conditions. After 3 h, the reference strain also starts to present PI positive cells that increase over time. Nevertheless a lethal effect of the colicin producing killer cells on the eukaryotic cancer sells can be assumed.

The initial results show that killing by E. coli producing colicin E1 is effective on eukaryotic cancer cells (MCF-7). Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used to stain DNA. It can be used to differentiate, apoptotic and normal cells, since it is membrane impermeant and generally excluded from viable cells. The high percentage of positive PI cells refers to cells that are permeable to it, thus, going into apoptosis. In this assay, after 2 h of incubation with the eukaryotic cells, the death rate was close to 40 % of the whole eukaryotic cell population. In comparison with the control conditions (MCF-7 alone + PI), where cells (maximum of 5%) due to trypsin treatment die, the effect is clearly visible. Furthermore, looking at the eukaryotic cell death after incubation with the reference strain (LuxR-receiver TOP 10), it can be suggested that the simple incubation of these mammalian cells with bacteria is causing cell death over time (maximum of 20% after 5 h). This effect is highly enhanced and faster when the bacteria produce colicin E1 (maximum of 37 % at 2 h). After 5 h, both bacterial strains produce similar results in killing the mammalian cancer cells. After reaching a peak, the killing efficiency drops, with a significant higher peak for E. coli bacteria producing colicin E1 than for the reference strain. On the other hand, using a stable cell line of MCF-7 expressing GFP, the effects of bacteria were not visible for the initial timepoints. The aim of this assay was to see the GFP signal droping down as E. coli progressed to kill the eukaryotic cells. Since GFP is a very robust protein we can suggest that colicin E1 producing bacteria start apoptotic events at early time (2 h, see Figure 8), but GFP loss can only be observed after a certain time delay (see Figure 9). For that reason, it would be necessary to observe also later timepoints to characterize the killing effect by GFP loss.

Figure 9: GFP expressing MCF-7 cells (%) over time. E coli bacteria producing colicin E1 protein cause GFP loss after 6 h, to an extend of 60% loss in comparison with the both control conditions that remained around 99 % GFP detection.

Summerized, the preliminary results suggest that colicin E1 producing E. coli bacteria might be able to kill cancer cells at a high rate. However, further studies need to provide more insight into these events, as it might be by other pro-apoptotic markers or by repeating this experiment using different conditions (timepoints, autoinducer concentrations, another eukaryotic cell type). Future studies should yield to information about the colicin E1 target in eukaryotic cells and perhaps answers how it can kill cancer cells and spare healthy ones.

Applications of BBa_K150009

This ColicinE1 Producer Controlled by 3OC6HSL Receiver Device sender was succesfully tested in an prey killer system with part BBa_K150000 of iGEM Team Heidelberg 2008 ([http://2008.igem.org/Team:Heidelberg/Project/Killing_II click for more details...]). (back)

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