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

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.

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.

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.

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.

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.

Conclusion

The fluorescence of several devices has been quantified according to a standardizable methods.
Taking part in an international consortium is always a great honor. This study was very enriching. It was a good opportunity to use a flow cytometer and to get used to it.
The results has been transmitted to the iGEM InterLab Measurement HQ. The last thing to do is waiting for the results.

Calibration

To obtain results in comparable units with the other iGEM teams, a conversion ratio according to the calibration run was first calculated:

The maximum fluorescence of each peak (i.e. at the center) was determined. Only the peaks with a variation coefficient (CV) under 5% are measured .
The MEFL value (given by the iGEM) for each RCP-30-5A peak is divided by the observed peak centers to produce a conversion ratio. The final conversion ratio is the average.

Protocols

Materials

All the DNA device used for this study were provided by the iGEM InterLab Measurement Kit.

The flow cytometer used for the study is a BD Accuri C6 Analysis with a laser at 488 nm and FL1-A filter at 533/30 nm. Calibration beads are RCP-30-5A (8 peaks) 3.0-3.4 µm. The cytometer was calibrated before the sample analysis. For each measurement, 10,000 events were acquired. Between 2 sample, washing runs were processed in order to avoid any contamination.

Protocols

Overnight cultures were diluted according to the iGEM normalization sheet, to an optical density of 0.02, but in LB (not TB)
Incubate 6 hours at 37°C
Measure OD₆₀₀ and dilute cells to obtain an OD₆₀₀ of 0.006 (106 cells.mL-1) required for the flow cytometer assays.
Adjust side-scatter (SSC) and forward scatter (FSC) PMT voltages using bacteria from your negative control, until the distribution of each is centered on the scale.
Adjust FITC/GFP PMT voltage using bacteria from your positive control, until the upper edge of the “bell curve” from the fluorescent population is one order of magnitude below the upper end of the scale
Acquire at least 10,000 events from a sample of calibration beads: RCP-30-5A (8 peaks), Rainbow Calibration Particles, 10⁷/mL, 3.0-3.4 µm.
Acquire at least 10,000 events for each biological sample
Divide the MEFL value for each RCP-30-5A peak by the observed peak centers, to produce a conversion ratio
Take the average of the conversion ratios: multiplying arbitrary units by this mean conversion ratio will change them into MEFL
Compute the geometric mean of fluorescence for each biological sample, excluding all events with values below 10
Multiply the geometric mean fluorescence for each sample by the mean conversion ratio, to produce a value in MEFL