Team:TU Eindhoven/Project/Characterization/Cell Viability

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.

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.

Bibliography