Interlab Measurement Study
The 2018 Queens_Canada iGEM team participated in iGEM’s Fifth Interlab Study. The objective of this year’s Interlab study was to standardize fluorescence measurements by avoiding the use of optical density through a more direct method of counting colony-forming units (CFUs). There can be a lot of variability in synthetic biology based on different instruments and techniques between labs, so this year’s study wanted to see if a more standardized and direct approach to measuring fluorescence could be obtained. In order to conduct the study, the team transformed the chemically competent DH5-alpha Escherichia coli with eight plasmids that was provided by iGEM in the distribution kits. We used the protocols provided by iGEM throughout the study.
First, we measured the OD600 of LUDOX CL-X in order to obtain a reference point to be used as a conversion factor to transform our absorbance (Abs600) data from the plate reader into a comparable OD600 measurement.
Next, the team conducted serial dilutions using silica microspheres that are similar in size and optical characteristics to E. coli to create a standard particle curve. As expected, the curve for the standard particles was linear.
Calibration 3: Fluorescein
Plate readers often report fluorescence values in arbitrary units that can vary between instruments, and thus labs. It is difficult to directly compare the measurements and values of fluorescence between instrument. Therefore, we created a standard fluorescence curve using fluorescein, a comparable fluorescent molecule to GFP. The serial dilution measurements of fluorescein were recorded in the same plate as the previous calibrations.
Cell Measurement Protocol
The cell measurements were done after the calibrations were completed. We transformed Escherichia coli DH5α with 8 plasmids provided in the iGEM Parts Distribution Kit plates. After the transformations, we plated the cells on chloramphenicol plates that were made in our lab. The next day, with many colonies to choose from, we picked 2 and inoculated them in LB medium and the appropriate amount of chloramphenicol. The liquid cultures were grown overnight in 37°C and 220 rpm. On Day 3, the overnight cultures were made into a 1:10 dilution using LB + chloramphenicol. The Abs600 was measured, and further diluted to an Absorbance of 0.02 in 12mL of LB+ Chloramphenicol. We covered out falcon tubes in aluminum foil to block the light. The samples were measured at 0 hours for fluorescence and Abs600, and again after 6 hours of addition growth.
Colony Forming Units per 0.1 OD600 E. coli cultures
The overnight cultures were diluted to OD600 in LB + Cam media. This was done in triplicate for each culture. The samples were further diluted using a serial dilution, to form three final dilutions of 8x104, 8x105, and 8x106. 100uL of the dilutions were each plated on chloramphenicol plates. The next day, the amount of colonies were counted by the team, and colony forming units were calculated.