Reliable and repeatable measurement is a key component to all engineering disciplines. However, the number of cells in the sample is a variability in measurements. The goal of the iGEM InterLab Study is to identify and correct the sources of systematic variability in synthetic biology measurements. This year, our team take part in the fifth InterLab study, using normalizing to absolute cell count or CFUs instead of OD to reduce variability of fluorescence measurements.

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

Methods

We followed this protocol to do the Interlab Study.
When we completed three of the calibration measurements, performing the cell measurements. Used the same plates, volumes and settings that we used in calibration protocol. We transformed E.coli DH5α competent cells with the 8 plasmids and picked 2 colonies from each of plates into 5 mL LB medium + Chloramphenicol. 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, 6 hours.

Result

Calibration
Calibration 1: OD600 reference point

Calibration 2: Particle Standard Curve

Cell measurement

Analyse

A standard curve in a linear relationship can be obtained by calibration experiments.
By comparing and analyzing the fluorescence values and Absorbance value of the different test devices at 0 and 6 hours, we can draw the following conclusions: the negative control and the positive control showed significant differences in the 6h fluorescence measurement results. Among the six different test equipment, 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 0.02, but there are errors with in the actual operation, can not be accurately diluted to 0.02. So we hope that the protocol can provide acceptable error range.
  2. In the second calibration, 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.

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

Team:FJNU-China/InterLab

Interlab