Overview

  In our project, the InterLab goal is to explore whether we can use standardized absolute cell counts or colony forming units (CFU) instead of OD to reduce lab-to-lab variability in fluorescence measurements.

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Materials

Strain used
Escherichia coli  DH5α

Plasmid used
Negative control: BBa_R0040 Plate 7 Well 2D
Positive control: BBa_I20270 Plate 7 Well 2B
Test Device 1: BBa_J364000 Plate 7 Well 2F
Test Device 2: BBa_J364001 Plate 7 Well 2H
Test Device 3: BBa_J364002 Plate 7 Well 2J
Test Device 4: BBa_J364007 Plate 7 Well 2L
Test Device 5: BBa_J364008 Plate 7 Well 2N
Test Device 6: BBa_J364009 Plate 7 Well 2P

Machine
Molecular Devices SpectraMax i3x

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Methods

  We followed this protocol to do the Interlab Study.
  All of the calibration measurements have been performed at least three independent experiments. We transformed 8 plasmids into E.coli DH5α competent cells, picked 2 colonies from each plate, and cultured the colonies into 5 mL LB medium supplemented with chloramphenicol when necessary. After culturing the cells overnight at 37°C and 220 rpm, we used plate reader to measure the Abs600 and fluorescence of samples at 0 and 6 hours.

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Result

Calibration
Calibration 1: OD600 reference point

Calibration 2: Particle Standard Curve

Cell measurement

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Analysis

  A standard curve in a linear relationship can be obtained by calibration experiments.
By comparing and analyzing the fluorescence values and absorbance values at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences of fluorescence values at 6 h. Among the six different test equipment, the device 4 has the strongest fluorescence, while device 3 has the lowest fluorescence.

Discussion

    During the experiment, we encountered two problems.
  1. In the cell measurement experiment, we need to dilute the cultures further to a target Abs600 of OD 0.02, but there are always errors in the actual operation. We cannot accurately dilute the solution to OD 0.02. So we hope that the protocol can provide acceptable error range.
  2. In the second calibration experiment, the particle standard curve log graph is not a straight line. We guess that it is due to pipetting error or the time to add the sample is too long.

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Reference

1.Beal J, Haddock-Angelli T, Gershater M, de Mora K, Lizarazo M, Hollenhorst J, et al. (2016) Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli. PLoS ONE 11(3): e0150182.
2.https://2018.igem.org/Measurement/InterLab
3.http://parts.igem.org/Part:BBa_J61002