Team:Edinburgh OG/InterLab
InterLab
Three broadly principles came up from Endy (2005) to identify synthetic biology: standardization, decoupling and abstraction. These have been adapted to promote the standardization of biological components and simplify and homogenize complicated biological problems. Among these three golden principles, standardization allows to achieve reliable and repeatable measurement, essential for experiment and result analysis of synthetic biology around the world. Over the past three years, iGEM committee successfully held “interlab” studies, to unify different units and data processing method. This will improve the measurement tools available to whole the iGEM and the synthetic biology community, finally establishing a baseline for replicability and reproducible of fluorescence measurements (GFP). This year, our team used a universal and detailed protocol and perform data analysis to quantify the expression of eight different reporter constructs. The measured Fluorescent Protein (GFP) was used as a proxy for different promoter activity.
For this study, we used the eight different constructs:
- Positive control (BBa_I20270)
- Negative control (BBa_R0040)
- Test Device 1: J23101+I13504 (BBa_J364000)
- Test Device 2: J23106+I13504 (BBa_J364001)
- Test Device 3: J23117+I13504 (BBa_J364002)
- Test Device 4: J23101.BCD2.E0040.B0015 (BBa_J364003)
- Test Device 5: J23106.BCD2.E0040.B0015 (BBa_J364004)
- Test Device 6: J23117.BCD2.E0040.B0015 (BBa_J364005)
1. Calibration
LUDOX-S40 was used as a single point reference to obtain a conversion factor that transforms Abs600 absorbance data into a standard OD600 measurement. A black with flat, 96-well plate was used with a column of 4 wells containing 100 µl 100% LUDOX-S40 and 4 wells containing 100 µl H2O. The absorbance 600 nm of all samples in all standard measurement modes of our plate reader was measured, and our Reference OD600 data was divided by our Abs600 data to get our correction factor. Data was converted to OD600 measurements through multiplication by our correction factor.
|
|
Reference spectrophotometer |
Microplate reader |
||
|
|
H2O |
LDX-HS40 100% |
H2O |
LDX-HS40 100% |
|
Replicate 1 |
0 |
0.043 |
0.224 |
0.206 |
|
Replicare2 |
0 |
0.043 |
0.189 |
0.195 |
|
Replicate 3 |
0 |
0.042 |
0.162 |
0.202 |
|
Replicate 4 |
0 |
0.042 |
0.231 |
0.212 |
|
Average |
0 |
0.0425 |
0.201 |
0.204 |
|
Corrected Abs600 |
|
0.0425 |
|
0.003 |
|
Reference OD 600 |
|
0.0425 |
|
0.0425 |
|
Correction factor |
|
1 |
|
14.1 |
2. Dilution series chart
We created 200 µl of 2.5 µM fluorescein stock solution and serially diluted it 1:2 with 1x PBS across plate columns 1-11. We then mixed 100 µl of fluorescein 5x stock solution with 100 µl of PBS in each well. Column 12 contained only 100 µl PBS buffer. This calibration plate was then measured in our plate reader under standard GFP settings and no path length correction to give us a standard measurement curve.
Cell Measurement Protocol
- Inoculate 2 colonies per DNA sample on 5-10 ml LB + Chloramphenicol. Grow 16-18 hours at 37˚C and 220 rpm
- Measure OD600 of overnight cultures
- Dilute cultures to a target OD600 of 0.02 in 10 ml 0.5x LB medium + Chloramphenicol and incubate at 37˚C and 220 rpm
- Take 1% of total volume (100 µl) samples at 0, 1, 2, 3, 4, 5, and 6 hours of incubation
- Place samples on ice and measure OD and Fl at the end of sampling point
