Difference between revisions of "Team:INSA-Lyon/Interlab"

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iGEM : Interlab

Interlab

Goals

This study aim to improve the tools available to both the iGEM community and the synthetic biology community.
The question to be answered is: How close can the values be when fluorescence is measured all around the world?

We decided to contribute to this exciting measurement experiment by comparing the activity of 3 engineered constitutive constructs in E. coli with the two iGEM HQ recommended protocols using either a plate reader or a flow cytometer.

Plate Reader

Measurements of both OD₆₀₀ and fluorescence were made in a plate Chameleon Idex 5. Transformation, inoculation procedures recommended by iGEM HQ were strictly followed.

Constructs fluorescence specific activity

The activity of each promoter was estimated by calculating the ratio fluorescence/OD₆₀₀ of the transformed strain.Optical density measured at 600nm gives an estimation of the number of the DH5alpha cell.

**OD₆₀₀:** All strains showed the same growth pattern except the strain carrying the device 1 that slightly impaired the bacterial growth:

**Fluorescence:** the strain transformed with device 3 showed no detectable activity (compare negative control and device 3) when device 2 is slightly less active than the positive control. Device 1 confers to the bacteria a very strong ability to fluoresce, well above the positive control:

**Specific activity of the 3 devices:** was obtained by dividing the fluorescence by the OD₆₀₀ for each strain. Device 1 confers a remarkable specific activity to the cells:

The positive control and device 2 show similar activities.
High variability in the measurements in the first hours of culture do not allowed to conclude a the fluorescence conferred by device 3.

Conclusion

Cells carrying the device 1 exhibits a spectacular fluorescence.

Calibration

OD₆₀₀

Add 100 μl LUDOX into wells A1, B1, C1, D1.
Add 100 μl of H2O into wells A2, B2, C2, D2
Measure absorbance 600 nm of all

This gives us a correction factor of 1.55, which has been used to compute the previous values.

FITC standard curve

Spin down FITC stock tube to make sure pellet is at the bottom of tube
Prepare 10x FITC stock solution by resuspending FITC in 1 mL of 1xPBS
Incubate the solution at 42°C for 4 hours
Dilute the 10x FITC stock solution in half with 1xPBS to make a 5x FITC solution and resulting concentration of FITC stock solution 2.5 μM
Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
Add 200 μl of FITC 5x stock solution into A1, B1, C1, D1
Transfer 100 μl of FITC stock solution from A1 into A2
Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
… repeat from A3 to A11 ...
Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste
Repeat dilution series for rows B, C, D
Measure fluorescence of all samples in all standard measurement modes in instrument

Protocols

Materials

Competent cells (Escherichia coli strain DH5α)
LB (Luria Bertani)
Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
Positive control: I20270
Negative control: R0040
Device 1: J23101+I13504
Device 2: J23106+I13504
Device 3: J23117+I13504

Protocols

Set your instrument to read OD₆₀₀ (as OD calibration setting)
Measure OD₆₀₀ of the overnight cultures
Dilute the cultures to a target OD₆₀₀ of 0.02
Incubate the cultures at 37°C and 220 rpm
Take 100 μL (1% of total volume) samples of the cultures at 0, 1, 2, 3, 4, 5, and 6 hours of incubation
Place samples on ice.
At the end of sampling point you need to measure your samples (OD and Fl measurement)

Flow Cytometry

Fluorescence measurement by flow cytometer

The fluorescence is analysed by the FL1-A channel (filter at 533/30 nm).
The following histograms analyses the intensity of fluorescence at 533 nm according to the cells count.

Fig. Fluorescence comparison between the positive control(red) and the negative control (black)

The fluorescence comparison between the positive and the negative control is significant. The peak of emission of the negative control has a lower intensity than the positive control.

The fluorescence of the each devices are analysed and computed together in order to visualise the difference between the 3 devices and the negative control.

Fig. Comparison between the 3 devices and the controls

The device 1 seems to induced the most intensive fluorescence, but the growth of the cells is limited. As hypothesis, the promoter may be too strong. A great amount of the cells ressources are dedicated to the production of GFP, that’s why the OD is so low.
The device 3 has a good growth according to the high OD. However the GFP production 300 times mower than the device 1. The promoter may be more weak. The device 2 is a good compromise between cells growth and GFP expression.

Reproducibility

In order to evaluate the reproducibility of the measurement, the replicates fluorescence profiles are superposed.

The curves are well superposed. The results are reproducible.

Further Analysis about Device 1

There is numerous cells with low fluorescence in the device 1. Is it contaminant or cells with very low GFP expression rate because of the too strong promoter? The forward scatter (FSC) and side scatter (SSC) provide several informations about the structure of the cells.

Structural analysis of device 1 *(The GFP producing cells are identified in green, the other ones are in red)*

The rate of GFP production is 68%. According to a structural view there is no significant difference. The ratio fluorescent/non fluorescent cells is conserved for similar cell size and granulometry. The hypothesis can not be validated.