Interlab Study
Our team elected to participate in iGEM’s international InterLab study “to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.” The efficacy of each gene’s expression was verified through fluorescence produced by the cell culture. The three genetic constructs being analyzed were J23101 + I13504, J23106 + I13504, and J23117 + I13504.
Study Protocol
We started overnight cultures from an individually picked colony and let grow for 18 hours in 17x100 mm Fisherbrand culture test tubes with approximately 6ml of LB media under appropriate antibiotics (Cm 34 µg/ml and Kan 50 µg/ml for respective plasmid constructs). Three separate cultures were created for each completed device. The cultures were grown in an Eppendorf New Brunswick, 12500 Series Incubating Platform Shaker at 220 rpm and 37.0 C. The optical density (OD) of each sample was determined, then samples were diluted to achieve an OD600 of 0.5. 200ul of each sample was pipetted into three separate wells (triplicate) of a 96-well black microplate. Three wells each were also used for a control (non-fluorescing E. coli, LB media, and empty blank wells. Samples were read at 485/20nm and 528/20nm with a BioTek Synergy 2 Multi-Mode Reader.
Optical density (OD600) was measured to obtain the same amount of cells per measurement. Green fluorescence was measured to determine fluorescence output of the cells.
Measured Quantities
OD600 does not have true units as it is a unitless ratio, but are reported in Absorbance Units (AU). There is no equivalent SI base unit for this measurement.
Fluorescence is reported in relative fluorescent units (RFU) and is a relative, unitless ratio. There is no equivalent SI base unit for this measurement.
Our spectrophotometer used to measure OD600 has a linear range of approximately 0.1-1.0 OD value. If values are above this range, we dilute our samples to conform to this range. Our spectrophotometer gives values to ten-thousandth (10-4), therefore we took the accuracy of the measurement to the thousandth (rounded the last decimal place up or down). Inside of this range, the precision does not vary significantly to our knowledge. A standard curve has been determined using known concentrations, which allowed us to find the linear range of our instrument.
The data were analyzed by using the control organism (E. coli) as a blank for each technical replicate in order to compensate for background fluorescence from the bacteria and from the LB media. In the chart presented below, the values are the raw data values with the respective blank value subtracted from them, and then averaged across the three biological triplicates.
