Team:IIT-Madras/Interlab study
Interlab Study
Motivation
Fluorescence measurements usually cannot be compared as they are reported in different units or being processed in different ways. Often we work around this by doing “relative expression” comparison. However, a lack of standard makes it harder to debug engineered biological constructs, harder to effectively share constructs between labs, and harder even to just interpret your experimental controls.
Experimental Design
Interlab study for 2016 iGEM aimed to quantify expression of five different reporter constructs which express GFP under the control of different promoter and RBS parts. The study focuses on the standardization of fluorescence measurements and primarily involves measurement of fluorescencea and OD600 values. It has been divided into 4 modules: OD600 reference point, FITC standard curve, Normalization, Cell measurement.
Instrumentation
We used Tecan Infinite M200 Pro instrument to measure OD600 and fluorescent values.
GFP and FITC sample, 100ul, readings were taken in NUN96ft Black Thermo plates. The instrument settings were kept as: Excitation/Emission wavelengths-481nm/511nm, Bandwidth-9/20, Gain-Manual(100), Flashes-25, Integration time-20.
OD600 sample, 100ul, measurements were taken in Tarsons 96 well micro test plate (Cat. No. 941196).
OD600 Reference Point
Ludox-S30 was taken as a single reference point to obtain a ratiometric conversion factor to transform the absorbance data into a standard OD600 measurement.
Results
Conclusion
