Stanford-Brown 2016

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.

Plate Reader Protocol

Calibration

Figure 1: Our team's FITC standard curve, as calculated by iGEM's official InterLab spreadsheet.

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 100 µl 100% LUDOX 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.

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

Figure 2: An illustration of the InterLab Study's protocol, done by team member Taylor Pullinger.

Data & Results

Figure 3: Our team's Abs₆₀₀ curve, as calculated by iGEM's official InterLab spreadsheet.

For our Abs₆₀₀ and OD₆₀₀ data, we used a Synergy HT plate reader and a 96-well plate. We used the following settings:
Wavelengths: 600
Pathlength correction: 977/900
Absorbance at 1 cm: 0.18

For our fluorescence data, we used a Synergy HT plate reader and a 96-well plate. We used the following settings:
Excitation: 485/20
Emission: 528/20
Optics: Top
Gain: 35
Read height: 1mm

Figure 4: Our team's fluorescence graph, made from our raw fluorescence data and calculated by iGEM's official InterLab spreadsheet.

Figure 5: Our team's Abs₆₀₀ data divided by our fluorescence data, calculated as an average with a standard deviation on a curve by iGEM's official InterLab spreadsheet.