Team:TU Eindhoven/Project/Characterization/Cell Viability

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Revision as of 17:59, 13 October 2014

iGEM Team TU Eindhoven 2014

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.

Table 1. The following tubes were prepared to incubate for 2 hours at 4°C in a shake incubator at 500 rpm.
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.

Table 2. Results of the analyze agar plates after incubation of ~16 hours.
Tube Name	Number of Cells Plated	Number of Cells 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%

Bibliography

iGEM Team TU Eindhoven 2014

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