Team:NAU-CHINA/InterLab

Template:2018_NAU-CHINA

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InterLab

Project

InterLab

Introduction

Reliable and repeatable measurement is a key component to all engineering disciplines. Based on this idea, the measurement committee encourages all competing teams to participate in the InterLab study. This year, the committee wants iGEMers from around the world to answer the following question together: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

Materials

1. Measurement Kit (provided with the iGEM distribution shipment) containing: 1mL LUDOX CL-X
150 μL Silica Bead (microsphere suspension)
Fluorescein (powder, in amber tube)
2. iGEM Parts Distribution Kit Plates
3. 1x PBS (phosphate buffered saline, pH 7.4 – 7.6)
4. ddH2O (ultrapure filtered or double distilled water)
5. Competent cells (Escherichia coli strain DH5α)
6. LB (Luria Bertani) media
7. Chloramphenicol (stock concentration 25 mg/mL dissolved in EtOH)
8. 50 mL Falcon tube (covered in foil to block light)
9. Incubator at 37°C
10. Plate reader (SpectraMax i3x)
11. 1.5 mL eppendorf tubes
12. Ice bucket with ice
13. Micropipettes (capable of pipetting a range of volumes between 1 μL and 1000 μL)
14. Micropipette tips
15. 96 well plates, black with clear flat bottom

Calibration

1. OD600 Reference point

We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform the absorbance (Abs600) data from our plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer.

Table 1. The data for OD600 measured by our plate reader, with a ratiometric conversion factor of 3.505.

2. Particle Standard Curve

We prepared a dilution series of monodisperse silica microspheres as instructed in the protocol.

Figure 1. Serial dilution of monodisperse silica microspheres.

The size and optical characteristics of these microspheres are similar to cells, and there is a known amount of particles per volume. This measurement allows us to construct a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells.

Table 2.The data for Abs600 measured by our plate reader.
Figure 2-1. Abs600 is plotted against the particle count.
Figure 2-2. Abs600 is plotted against the particle count on a logarithmic scale.
Table 3. The data for particles/Abs600.
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