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 the time. Top right panel: Optical densities of prey cells treated with killer cells plotted against the 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 the 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.

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).

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.

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...]).

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