Team:INSA-Lyon/Interlab
Interlab Study
Goals
This study aims at improving 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.
Comparison of the fluorescence specific activity of all devices
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 DH5α cell.
Positive control and device 2 show similar activities.
High variability in the measurements in the first hours of culture did not allow to conclude the fluorescence ability conferred by device 3.
Conclusion
Cells carrying device 1 exhibit a spectacular fluorescence.
Calibration
OD600
Add 100 μl LUDOX into wells A1, B1, C1, D1.
Add 100 μl of H
This gave us a correction factor of 1.55, which was 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 cytometry
The fluorescence was analyzed by the FL1-A channel (filter at 533/30 nm).
The following histograms show analysis of the intensity of fluorescence at 533 nm according to the cells count.
The fluorescence comparison between the positive and the negative control was significant. The peak of emission of the negative control had a lower intensity than the positive control.
Fluorescence levels of each device were analyzed and computed together in order to visualize the difference between the 3 devices and the negative control.
Device 1 seemed to induce the most intensive fluorescence, but the growth of the cells was limited.
As a hypothesis, the promoter may be too strong.
A great amount of the cells resources might be dedicated to the production of GFP, which would explain why the OD is so low.
Device 3 showed a good growth according to the high OD. However the GFP production was 300 times lower than the device 1.
The promoter may be weaker.
Device 2 is a good compromise between cell growth and GFP expression.
Reproducibility
In order to evaluate the reproducibility of the measurement, fluorescence profiles replicates were overlapped.
The curves overlapped nicely. The results are reproducible.
Further Analysis about Device 1
There were numerous cells with low fluorescence in the device 1. Was it due to the presence of contaminants or cells with very low GFP expression rate because of the too strong promoter? The forward scatter (FSC) and side scatter (SSC) provided several information about the structure of the cells.
The proportion of cells producing GFP 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 was quantified according to standardizable methods.
Taking part in an international consortium is always a great honor. This study was very enriching.
It was a good opportunity to learn how to use a flow cytometer.
Our results were sent to the iGEM InterLab Measurement HQ. Now, we can't wait to discover the results of this meta-analysis, and to see the 2016 iGEM teams's works valued in next year's publication !
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 peaks with a variation coefficient (CV) under 5% were measured .
The MEFL value (given by the iGEM) for each RCP-30-5A peak was divided by the observed peak centers to produce a conversion ratio.
The final conversion ratio is the average.
Protocols
Materials
All DNA devices used for this study were provided in 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 samples, washing runs were performed 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.
