Calibration
LUDOX-S30 was used as a single point reference to obtain a conversion factor that transforms Abs600 absorbance data into a standard OD600 measurement. A 96-well plate was prepared with a column of 4 wells containing 100µl 100% LUDOX and 4 wells containing 100µl H2O. The absorbance 600nm 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.
Team:Stanford-Brown/SB16 Collaborations Interlab
2016 InterLab Measurement Study
Background
For the past two years, iGEM has hosted the InterLab Measurement Study, utilizing iGEM's resources and spirit of collaboration to create the two biggest interlaboratory studies ever done in synthetic biology. These studies seek to standardize the tools available to the synthetic biology community and focus on establishing a baseline for replicability of fluorescence measurements. This year, iGEM teams from around the world tested two protocols that used plate readers and flow cytometry to quantify the expression of five different reporter constructs. The measured fluorescence of Green Fluorescent Protein (GFP) was used as a proxy for promoter activity.
For this study, we used the following constructs:
• J23101 + I13504 as Test Device 1
• J23106 + I13504 as Test Device 2
• J23117 + 13504 as Test Device 3
• I20270 as our Positive Control Device
• R0040 as our Negative Control Device
All devices and parts were obtained from the iGEM 2016 InterLab Distribution Kit. Devices were made with a pSB1C3 plasmid backbone and transformed in the E. coli strain NEB5-alpha.
For the past two years, iGEM has hosted the InterLab Measurement Study, utilizing iGEM's resources and spirit of collaboration to create the two biggest interlaboratory studies ever done in synthetic biology. These studies seek to standardize the tools available to the synthetic biology community and focus on establishing a baseline for replicability of fluorescence measurements. This year, iGEM teams from around the world tested two protocols that used plate readers and flow cytometry to quantify the expression of five different reporter constructs. The measured fluorescence of Green Fluorescent Protein (GFP) was used as a proxy for promoter activity.
For this study, we used the following constructs:
• J23101 + I13504 as Test Device 1
• J23106 + I13504 as Test Device 2
• J23117 + 13504 as Test Device 3
• I20270 as our Positive Control Device
• R0040 as our Negative Control Device
All devices and parts were obtained from the iGEM 2016 InterLab Distribution Kit. Devices were made with a pSB1C3 plasmid backbone and transformed in the E. coli strain NEB5-alpha.
Plate Reader Protocol
200µl 2.5µM fluorescein stock solution was created and serially diluted 1:2 with 1x PBS across plate columns 1-11. 100µl of fluorescein 5x stock solution were mixed in with 100µl of PBS in each well. Column 12 contained only 100µl 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
1. Inoculate 2 colonies per DNA sample on 5-10ml LB + Chloramphenicol. Grow 16-18 hours at 37˚C and 220rpm
2. Measure OD600 of overnight cultures
3. Dilute cultures to a target OD600 of 0.02 in 10ml 0.5x LB medium + Chloramphenicol and incubate at 37˚C and 220rpm
4. Take 1% of total volume (100µl) samples at 0, 1, 2, 3, 4, 5, and 6 hours of incubation
5. Place samples on ice and measure OD and Fl at the end of sampling point
Cell Measurement Protocol
1. Inoculate 2 colonies per DNA sample on 5-10ml LB + Chloramphenicol. Grow 16-18 hours at 37˚C and 220rpm
2. Measure OD600 of overnight cultures
3. Dilute cultures to a target OD600 of 0.02 in 10ml 0.5x LB medium + Chloramphenicol and incubate at 37˚C and 220rpm
4. Take 1% of total volume (100µl) samples at 0, 1, 2, 3, 4, 5, and 6 hours of incubation
5. Place samples on ice and measure OD and Fl at the end of sampling point
Data & Results
