Team:UNC-Asheville/InterLab
Interlab Measurement Assay
We didn't break the expensive machine!
Overview
Variability in the processing of data and repeatability of assays poses a challenge in the field of synthetic biology. The interlab measurement allows for the establishment of a baseline for replicability of fluorescence measurements. GFP is a commonly used biomarker, and studying how these RBS devices affect florescence, using hundreds of independent lab results, is a big step towards standardizing these parts.
Results
The Protocol
LUDOX-S40 was used to obtain a conversion factor to normalize the absorbance measurements to a standard optical density of 600 nanometers (OD 600). The correction factor was determined by measuring LUDOX-S40 in the multimode plate reader Synergy HTX (BioTek, Software gen5 2.09), and comparing the subsequent data to the optical density reference point. To normalize the fluorescence measurements, the fluorescence readings were obtained from a serial dilution of fluorescein in phosphate buffered saline solution (PBS).
A dilution series of fluorescein in 4 replicates was created, and the fluorescence was quantified in a 96 well plate (Grenier Half Area Flat Bottom). The resulting data was used to correct the cell based readings to an equivalent fluorescein concentration, and subsequently converted to a concentration of GFP. The creation of a standard curve for the correction factor and fluorescence measurements allowed for the direct comparison of data between labs.
The E. coli strain K-12 DH5-alpha was used for the calibration measurements. Two colonies were chosen from the performed transformation and inoculated in 5 mL cultures of LB and chloramphenicol. Both strains were incubated overnight at 37℃ and 220 rpm. The OD600 was measured for both of the overnight cultures.
Each culture was diluted to a target OD600 of 0.02 in a 12 ml medium of LB with chloramphenicol. The diluted cultures were incubated at 37°C and 220 rpm. 500 µL of each culture was taken at 0, 2, 4, and 6 hours of incubation. The postliminary data was used to create the calibration curve.
