Team:NKU CHINA/interlab
Overview
Poverty in taking reliable and repeatable measurements remains a key obstacle in establishing synthetic biology as an engineering discipline. The Measurement Committee has been studying the measurement procedure for green fluorescent protein (GFP) over the last several years by interlab. The most commonly used markers though GFP is in synthetic biology, labs often resort to making relative comparisons, which makes it difficult for labs to share and data and/or constructs.
The goal of the fifth iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements by answering the question "Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD? "
As we know in the previous study, the fluorescence value measured by a plate reader is an aggregate measurement of an entire population of cells, we need to divide the total fluorescence by the number of cells in order to determine the mean expression level of GFP per cell. Due to the fact that the "optical density (OD)" of the sample is an approximation of the number of cells varying from lab to lab, we decided to use a special silica beads that are roughly the same size and shape as a typical E. coli cell to set up a universal, standard "equivalent concentration of beads" measurement.
Materials
- Competent cells (Escherichia coli strain DH5α)
- 1 mL LUDOX CL-X (provided in kit)
- 300 µL Silica beads - Microsphere suspension (provided in kit, 4.7 x 108 microspheres)
- LB (Luria Bertani) media
- Fluorescein (provided in kit)
- 10 mL 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
- Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
- 50 mL Falcon tube (or equivalent, preferably amber or covered in foil to block light)
- Incubator at 37°C
- 1.5 mL eppendorf tubes for sample storage
- Ice bucket with ice
- Micropipettes and tips
- 96 well plate, black with clear flat bottom preferred (provided by team)
From Distribution Kit, all in pSB1C3 backbone:
- Negative control BBa_R0040
- Positive control BBa_I20270
- Test Device 1 BBa_J364000
- Test Device 2 BBa_J364001
- Test Device 3 BBa_J364002
- Test Device 4 BBa_J364007
- Test Device 5 BBa_J364008
- Test Device 6 BBa_J364009
Methods
- Add 100 µL LUDOX into wells A1, B1, C1, D1
- Add 100 µL of dd H2O into wells A2, B2, C2, D2
- Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements
- Record the data in the table below or in your notebook
- Import data into Excel sheet provided (OD600 reference point tab)
- Obtain the tube labeled "Silica Beads" from the InterLab test kit and vortex vigorously for 30 seconds
- Immediately pipet 96 µL microspheres into a 1.5 mL eppendorf tube
- Add 904 µL of ddH2O to the microspheres
- Vortex well. This is your Microsphere Stock Solution
- Repeat dilution series for rows B, C, D
- Re-Mix (Pipette up and down) each row of plate immediately before putting in the plate reader
- Measure Abs600 of all samples in instrument
- Record the data in your notebook
- Import data into Excel sheet provided (particle standard curve tab)
- Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
- Prepare 10x fluorescein stock solution (100 µM) by resuspending fluorescein in 1 mL of 1xPBS
- Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 µM: 100 µL of 10x fluorescein stock into 900 µL 1xPBS
- Add 100 µL of PBS into wells A2, B2, C2, D2...A12, B12, C12, D12
- Add 200 µL of fluorescein 1x stock solution into A1, B1, C1, D1
- Transfer 100 µL of fluorescein stock solution from A1 into A2
- Mix A2 by pipetting up and down 3x and transfer 100 µL into A3...
- Mix A3 by pipetting up and down 3x and transfer 100 µL into A4...
- Mix A4 by pipetting up and down 3x and transfer 100 µL into A5...
- Mix A5 by pipetting up and down 3x and transfer 100 µL into A6...
- Mix A6 by pipetting up and down 3x and transfer 100 µL into A7...
- Mix A7 by pipetting up and down 3x and transfer 100 µL into A8...
- Mix A8 by pipetting up and down 3x and transfer 100 µL into A9...
- Mix A9 by pipetting up and down 3x and transfer 100 µL into A10...
- Mix A10 by pipetting up and down 3x and transfer 100 µL into 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 instrument
- Record the data in your notebook
- Import data into Excel sheet provided (fluorescein standard curve tab)
- Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH2O
- Thaw competent cells on ice
- Pipette 50 µL of competent cells into 1.5 mL tube
- Pipette 1 µL of resuspended DNA into 1.5 mL tube
- Pipette 1 µL of control DNA into 2 mL tube
- Close 1.5 mL tubes, incubate on ice for 30 min
- Heat shock tubes at 42°C for 45 sec
- Incubate on ice for 5 min
- Pipette 950 µL SOC media to each transformation
- Incubate at 37°C for 1 hours, shaking at 200-300 rpm
- Pipette 100 µL of each transformation onto petri plates
- Spin down cells at 6800 g for 3 min and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates
- Incubate transformations overnight (14-18 hr) at 37°C
- Pick single colonies for PCR
- Count colonies for control transformation
- Pick 2 colonies from each of the transformation plates and inoculate in 5-10 mL LB medium + Chloramphenicol. Grow the cells overnight (16-18 hours) at 37°C and 220 rpm
- Make a 1:10 dilution of each overnight culture in LB + Chloramphenicol (0.5 mL of culture into 4.5 mL of LB + Chlor)
- Measure Abs600 of these 1:10 diluted cultures
- Record the data in your notebook
- Dilute the cultures further to a target Abs600 of 0.02 in a final volume of 12 mL LB medium + Chloramphenicol in 50 mL falcon tube (amber, or covered with foil to block light)
- Take 500 µL samples of the diluted cultures at 0 hours into 1.5 mL Eppendorf tubes, prior to incubation. (At each time point 0 hours and 6 hours, you will take a sample from each of the 8 devices, two colonies per device, for a total of 16 Eppendorf tubes with 500 µL samples per time point, 32 samples total). Place the samples on ice
- Incubate the remainder of the cultures at 37°C and 220 rpm for 6 hours
- Take 500 µL samples of the cultures at 6 hours of incubation into 1.5 mL Eppendorf tubes. Place samples on ice
- At the end of sampling point you need to measure your samples (Abs600 and fluorescence measurement), see the below for details
- Record data in your notebook
- Import data into Excel sheet provided (fluorescence measurement tab)
- Culture colonies for two Positive Control (BBa_I20270) cultures and your two Negative Control (BBa_R0040) cultures for 16-18 hours
- Dilute the overnight culture to OD600 = 0.1 in 1 mL of LB + Cam media. Do this in triplicate for each culture. Check the OD600 and make sure it is 0.1
- Aseptically spead plate 100 µL on LB + Cam plates for those Final Dilution Factor is 8 x 104 or 8 x 105 or 8 x 106
- Incubate at 37°C overnight and count colonies after 18-20 hours of growth
- Count the colonies on each plate with fewer than 300 colonies. Multiple the colony count by the Final Dilution Factor on each plate
Results
According to the Reference OD600, We calculate the final result:
OD600/Abs600=3.818
All cell density readings using this instrument with the same settings and volume can be converted to OD600, so that we can use this ratio to convert subsequent experimental data.
We prepare a dilution series of monodisperse silica microspheres and measure the Abs600 in plate reader. The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.
The final result:
mean of med-high levels=6.24E+08
We prepare a dilution series of fluorescein in four replicates and measure the fluorescence in a 96 well plate in plate reader. By measuring these we generate a standard curve of fluorescence for fluorescein concentration. We will be able to use this to convert our cell based readings to an equivalent fluorescein concentration.
Final results:
Mean µM fluorescein / a.u.=3.60E-05
MEFL / a.u.=2.17E+08
