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)
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 PLuxR 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)
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 PLuxR 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)
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)
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).
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)
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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