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Team:TU Eindhoven/Project/Characterization/Cell Viability
From 2014.igem.org
Cell Viability Assay
Because of the troubles with the previous viability assay another option was chosen to check the viability of the bacteria. Basically, all steps are performed for the click reaction on the cells and those cells are plated in specific amounts on agar plates and incubated for ~16 hours to check if the bacteria still grew. This assay is described in the following protocol.
First, protein expression is induced as described in the protocol for Protein Expression. However instead of PBS-BSA 0.1% just plain PBS is used to resuspend the cells. Two different batches were tested in this assay to ensure that the outcomes are reliable if they match each other.
For the first reaction, the cells are incubated in a solution of 30 µM DBCO-PEG-10 kDa. Previous experiments have shown that the click reaction on the cell membrane occurs, this confirms that in this solution DBCO-PEG-10 kDa will attach to the cell membrane and therefore is not tested again.
For the second reaction, the cells are incubated in a 30 µM solution of PEG 3350 Dalton. These PEG molecules do not have the DBCO group to click onto the cell membrane. However they have the same influence on the environment in which the cells react. This second reaction was done to simulate the environment in which the click reaction occurs. The only difference between the cells is that in the first case the PEG molecules are covalently bound to the cell membrane, which can have a negative effect on the cell viability. See Protocol cell viability
| Tube Name | [DBCO and PEG no DBCO] | Cells [10^9 mL^-1] | DBCO 10 kDa (5 mM) | PEG no DBCO 3350 Da (5 mM) |
|---|---|---|---|---|
| DBCO 1 | 30 µM | 200 µL | 1.21 µL | 0 µL |
| DBCO 2 | 30 µM | 200 µL | 1.21 µL | 0 µL |
| PEG 1 | 30 µM | 200 µL | 0 µL | 1.21 µL |
| PEG 2 | 30 µM | 200 µL | 0 µL | 1.21 µL |
Meanwhile, 12 agar plates were prepared with kanamycin and chloramphenicol to plate the cells.
After the incubation, the samples are diluted to the required concentration of cells before plating. Concentrations of cells needed for these plates are approximately 1000, 100 and 10 cells. For this the following dilution series were used.
After diluting, 100 µL of each sample was plated on an agar plate and incubated for ~16 hours at 37°C.
The next day the plates were analyzed. For every plate the colonies were marked manually and counted. The results are shown in the table below.
| Tube Name | Number of Cells Plated | Number of Colonies Marked | % Survival |
|---|---|---|---|
| Dilution DBCO 1_2 | 1000 | 321 | 32.1% |
| Dilution DBCO 1_3 | 100 | 27 | 27% |
| Dilution DBCO 1_4 | 10 | 2 | 20% |
| Dilution DBCO 2_2 | 1000 | 343 | 34.3% |
| Dilution DBCO 2_3 | 100 | 43 | 43% |
| Dilution DBCO 2_4 | 10 | 6 | 60% |
| Avarage DBCO | 30% | ||
| Dilution PEG 1_2 | 1000 | 355 | 35.5% |
| Dilution PEG 1_3 | 100 | 38 | 38% |
| Dilution PEG 1_4 | 10 | 1 | 10% |
| Dilution PEG 1_2 | 1000 | 523 | 52.3% |
| Dilution PEG 1_3 | 100 | 42 | 42% |
| Dilution PEG 1_2 | 10 | 4 | 40% |
| Average PEG | 36% |
In this case it is obvious that a part of the cells is still viable in both the reactions. As seen in the results in table 2 there is a small difference between the viability of the cells clicked with DBCO-PEG compared to those without this molecule. However based on the small amount of data and the method of registering the colonies more research is needed to confirm this difference.
With this assay it is confirmed that the membrane can take the stress caused by the molecules clicked on the membrane and are able to survive. It can be concluded that the cells are viable after protein expression and the click-reaction. However, it is not known in which way this specific environment influences the cell viability. Due to the variety of the molecules which can be used for the click reaction the environment changes for every reaction. To check the viability of the cells in specific environments these assay have to be performed again.
