Team:William and Mary/Interlab
Interlab Measurement Study
Introduction
Inter-laboratory studies have great implications in both academia research and industry. Comparison of results can not only help determine the characteristics of certain products, but can also validate the test method and determine the source of uncertainty. Synthetic biology aims to achieve predicable gene expression outcomes [1], but challenges for this goal still exist on every level from parts design, circuity complexity to measurement methods. iGEM InterLab study is exactly designed to unravel the source of unpredictability and to quantify the degree of variability [2], the logical of which William and Mary iGEM team shares deeply. We have been an active participator of the InterLab Study since 2015 (the second year William and Mary joined the iGEM family) and we are very honored to be able to continue to contribute this study.
This year, the objective of InterLab is to test the precision of gene expression over different RBS devices with a GFP reporter. Teams from around the world are using the standard biological parts, same laboratory bacterium and standardized measurement procedure provided in a detailed protocol. Our team was excited about this year’s project and the improvements that InterLab has made such as the dried down DNA and extra reagents. We started our study on August, 8th.
Methods
Transformation
We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5-alpha cells. Colonies were given 16 hours to grow.
Calibration
Before we started plate reader measurement, we obtained the OD600 reference point and the fluorescein fluorescence curve in the microplate reader to standardize the absorbance reading and cell-based fluorescence reading. Our model was a Synergy H1 Hybrid Multi-Mode Microplate Reader. Ludox-S40 silica nanoparticles were used to calculate the correction factor of OD600. Black 96-well plates with clear bottoms were used. For the plate reader our excitation and emission setting were 485 nm and 528 nm respectively (Same setting was used for all experiments below).
The dilution curve of fluorescein was performed by carrying out a 11-step, 2-fold serial dilution of green fluorescein. Final scaling level was determined from medium-high points in the dilution that is likely to be less impacted by saturation or pipetting error. The μM Fluorescein/a.u.is defined as mean of mid-high level fluorescein concentration divided by the obtained plate reader reading.
Cell Measurement
2 colonies of each device was inoculated over night into 5 ml Luria- Bertani medium with 25 μg/mL Chloramphenicol in a 37°C, 220 rpm shaking incubator. Cell cultures were diluted to a target OD600 of 0.02 into same LB medium in 50 mL falcon tube covered with foil before use Diluted cultures were further grown at 37°C and 220 rpm. At 0, 2, 4, and 6 hours of incubation, 500 μL aliquot was taken from each two colonies of the 8 devices and were placed immediately on ice to prevent further growth. At the end of sampling point, 4 replicates 100 μl of each sample was pipetted into a 96-well microplate with the arrangement as below. Data were imported into the Excel Sheet for submission (William_and_Mary Measurement spreadsheet).