Team:Utah State/InterlabStudy

Introduction

Our team elected to participate in iGEM’s first international interlab study “to obtain fluorescence data for three specific genetic devices expressing GFP from iGEM teams around the world.” The efficacy of each gene’s expression was verified through fluorescence produced by the cell culture. The data we collected was sufficient and accepted by iGEM. Results from every participating team will be recognized at the convention.

Study Protocol

We started overnight cultures from an individually picked colony and let grow for 16 hours in 14ml culture tubes (approx. 6ml culture volume) of LB media under appropriate antibiotics (Cm 34ug/ml and Kan 50ug/ml for respective plasmid constructs). The optical density (OD) of each sample was determined, then samples were diluted to achieve an OD600 of 0.5 and resuspended in 0.15M NaCl solution to eliminate the background fluorescence seen by LB media. 200ul of each sample was pipetted into three separate wells (triplicate) of a 96-well black microplate. Three wells each were also used for a control (non-fluorescing E. coli), 0.15M NaCl saline solution, and empty blank wells. Samples were read at 360nm and 528nm.

Controls used were XL1-Blue E. coli (same strain used for fluorescence studies), 0.15M NaCl (resuspension solution for the E. coli), empty blank wells.

Optical density (OD600) was measured to obtain the same amount of cells per measurement. Green fluorescence was measured to determine fluorescence output of the cells.

Our first measurement was done with E. coli resuspended in LB media (approximately 20-25 minutes to prep and measure) but we realized that the background fluorescence of the LB media clouded our measurements. We then performed the prep and measurement using 0.15M NaCl solution to resuspend cells. This second prep and measurement took about 20 minutes.

Because the microplate reader was already available in our research laboratory, the costs to acquire a set of measurements is minimal. We approximate that the non-reusuable components would cost less than $1.00 total cost (pipette tips, LB media, 0.15M NaCl solution, appropriate antibiotics).

Measured Quantities

OD600 does not have true units as it is a unitless ratio, but are reported in Absorbance Units (AU). There is no equivalent SI base unit for this measurement.

Fluorescence is reported in relative fluorescent units (RFU) and is a relative, unitless ratio. There is no equivalent SI base unit for this measurement.

Our spectrophotometer used to measure OD600 has a linear range of approximately 0.1-1.0 OD value. If values are above this range, we dilute our samples to conform to this range. Our spectrophotometer gives values to ten-thousandth (10-4), therefore we took the accuracy of the measurement to the thousandth (rounded the last decimal place up or down). Inside of this range, the precision does not vary significantly to our knowledge. A standard curve has been determined using known concentrations, which allowed us to find the linear range of our instrument.