The Fifth International InterLaboratory Measurement Study is one of the largest collaborative studies ever performed in synthetic biology, featuring the work of iGEM teams across the world. Each annual iteration of the study is dedicated to identifying and rectifying sources of variability in measurements and protocols across lab settings. This year’s edition primarily focused on standardizing absorbance and fluorescence measurements of GFP-expressing bacteria, correcting for human and machine variability across labs.

Per the iGEM protocol, we transformed New England Biosciences 5-alpha E. coli with the eight following plasmids from the iGEM Distribution Kit 7:

Positive Control: BBa_I20270
Negative Control: BBa_R0040
Test Device 1: BBa_J364000
Test Device 2: BBa_J364001
Test Device 3: BBa_J364002
Test Device 4: BBa_J364007
Test Device 5: BBa_J364008
Test Device 6: BBa_J364009

Bacterial colonies were grown either in liquid cultures or plated with Luria Bertani (LB) broth and chloramphenicol; Each plasmid backbone confers bacterial resistance to chloramphenicol (pSB1C3 backbone).

Calibration: 3 Experiments

1. OD600 reference point

The purpose of the first calibration is to establish a conversion from “absorbance at 600nm” (Abs600) data in our 96 well plates into an “optical density at 600nm” (OD600) measurement, as would be obtained in a standard spectrophotometer. “Such conversion is necessary because plate reader measurements of absorbance are volume dependent; the depth of the fluid in the well defines the path length of the light passing through the sample, which can vary slightly from well to well.” To do so, we first measured the absorbance values of LUDOX-S30 versus deionized H2O. Reading the LUDOX-S30 absorbance value from our spectrophotometer, we subtract the dH2O absorbance to get a corrected absorbance value. Then, the LUDOX-S30 OD600 from iGEM’s reference spectrophotometer is divided by our corrected absorbance value to give a standardized conversion factor.

Figure 1: Raw Absorbance Values and Correction Factor. Calculated with iGEM Interlab Spreadsheet.

2. Particle Standard Curve

Our silica microspheres have similar size and optical characteristics as cells but can be mass produced and distributed globally so that all labs will have the same size and concentration of microspheres. Measuring the absorbance of these beads allowed our lab to construct a standard curve of particle concentration (seen below), “which can be used to convert Abs 600 measurements to an estimated number of cells.”

Figure 2: Microsphere Solution Serial Dilution Protocol

Per the protocol given by iGEM, we prepared out microsphere stock solution and serially diluted this solution over 11 wells each with a total of 100ul. Throughout this process we mixed by pipetvting up and down 3 times before each transfer of solution, as well as one final remixing before measurement by the plate reader.

3. Particle Standard Curve

In order to standardize our fluorescence data, we used iGEM-provided fluorescein (a small, inexpensive molecule with similar excitation properties to GFP), to “generate a standard curve of fluorescence for fluorescein concentration.” We followed the iGEM protocol and prepared the fluorescein stock solution via serial dilution, similar to the procedure for creating the microsphere absorbance standard curve.

The resulting fluorescence curve could be used to back-calculate cell-concentration and GFP expression in an unknown plate or liquid culture.

Cell Measurement

Protocol: Cell measurement

Transform Escherichia coli DH5α with specified plasmids and grow overnight on plates.
Pick 2 colonies from each plate and grow these overnight in seperate liquid cultures.
Dilute cultures to an Abs600 value of 0.02 in a final volume of 12 ml of LB and our antibiotic, chloramphenicol.
Take 500ul of each sample into seperate eppendorf tubes and place on ice.
Incubate original 12ml solution for 6 hours more.
Take 500ul from from each of the individual incubated samples and pipette into individual eppendorf tubes.
Check the Abs600 and fluorescence of all eppendorfs, by pipetting replicates, diluting on a 96 well plate, and recording data.

Protocol: Colony Forming Units per 0.1 OD600 E. coli cultures

Create dilute replicate solutions of our positive and negative control cultures makings sure that the initial optical density of each is close to .1 OD600.
Grow these cultures overnight and dilute further to final dilution factors of of 8x10^4, 8x10^5 and 8x10^6 and innoculate onto plates.
Count colonies on all plates.

Team:Stanford-Brown-RISD/InterLab