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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.
| 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 |
