Background

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:

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.