In the Interlab study all 2018 iGEM teams are invited to participate at the International InterLaboratory Measurement Study in synthetic biology. The characterization of standard biological parts is a fundamental ability of synthetic biology. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements by measuring the fluorescence of GFP expression.
This year, we want your help in answering the following 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?
We complete the calibration before we started with the cell measurement.
We used LUDOX CL-X as a single point reference to obtain a conversion factor to transform our absorbance (Abs620) data from our plate reader into a comparable OD620 measurement as would be obtained in a spectrophotometer. We measured the absorbance at 620 nm from LUDOX Cl-X and H2O.
The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allowed us to construct a standard curve of particle concentrations which can be used to convert Abs620 measurement to an estimated number of cells. We used silica beads – Microsphere solution and measured different solutions to construct the standard curve.
The absolute fluorescence values cannot be directly compared from one instrument to another. Because of this it is necessary for each team to create a standard fluorescence curve. We prepared a dilution series of fluorescein in four replicates and measured the fluorescence (485nm, 535nm) with the plate reader.
We used competent E. coli K-12 DH5-alpha cells to transform the devices from the Distribution Kit. All plasmids are in pSB1C3 backbone with a chloramphenicol resistance. The samples consist of one negative, one positive control and the six test devices. After the transformation we picked two colonies from each transformation plate and incubated it in LB medium with chloramphenicol overnight.
We measured Abs620 of these 1:10 diluted cultures and dilute the cultures further to a target Abs620 of 0.02. Thereafter, two samples per culture were prepared. One at hour 0 and another incubated at 37 ° C and 220 rpm for 6 hours.
Layout for the Abs620 and Fluorescence measurement: We had two plates, one for each point 0 and 6 hour and read fluorescence (485nm, 535nm) and absorbance (620 nm).
|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|
Colony Forming Units (CFU)
We used this calibrate OD600 to colony forming unit counts, which are directly relatable to the cell concentration of the culture. The samples consist of two colonies from the negative control and two from the positive control. For the measurement we made dilution series from the overnight cultures. After the colonies grew, we counted all colonies on the plates with less than 300 colonies and multiply the colony count by the final dilution factor on each plate.
In total, the Fluorescence per OD increases from hour 0 to hour 6.
Device 3 is a non-fluorescent sample.
Device 1 and 4 have the highest fluorescence.
Fluorescence depends on the number of particles and thus on the expression of the bacterial colonies and thus, also on the bacterial density.
The graphs show the Fluorescence per Particle of the individual devices.
Equipment and Settings
Plate reader: Tecan infinite 200 Pro
Application: Tecan i-control
Plate: Greiner 96 Flat Bottom Transparent Polystyrol
Absorbance: 620 nm
Fluorescence: Excitation Wavelength: 485 nm
Emission Wavelength: 535 nm