Objectives

Synthetic biology, also called engineering biology, differentiates itself from the field of biology in general through its ability to repeat and reproduce measurements and results. This reproducibility is apparent across all other engineering disciplines as well, and aids researchers in making effective comparisons for interpreting experimental controls and debugging engineered biological constructs. Through Interlab Study, iGEM’s Measurement Committee aims to achieve such reproducibility for the green fluorescent protein (GFP) in particular by developing a robust and detailed measurement protocol that anyone can follow.

In Interlab 2018, iGEM aims to examine if it is possible to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD. For this, we were required to measure the cell density of Escherichia coli DH5⍺ cells using two methods: by converting between absorbance of cells to the absorbance of a known concentration of beads, and by counting colony-forming units (CFUs) from the sample.

Overview

In the first method, silica beads modelled after (roughly the same shape and size of) a typical E. coli cell are used to estimate the actual amount of E. coli cells during the fluorescence measurement of the cells. In this method, silica beads were made to model a typical E. coli cell’s light scattering. As a sample of these silica beads gives a consistent and known absorbance measurement at 600 nm, absorbance measurements from a sample’s cell density can be converted into an “equivalent concentration of beads” measurement that should be more universal and comparable between different labs.

Another way of approximating cell concentration in a sample of bacterial culture is by plating a known volume of the sample and letting colonies grow. As each bacterial colony is assumed to represent a single cell (for cells that do not stick together), the cell concentration in the sample is then directly proportional to the number of CFUs. Using a scaling factor computed from negative and positive control CFUs, the absorbance measurements can be converted to CFU.

Parts Received