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.

2. Particle Standard Curve

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

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.

3. Fluorescence standard curve

We prepared a dilution series of fluorescein as instructed in the protocol.

By measuring the serial dilutions in our plate reader, we generated a standard curve of fluorescence for fluorescein concentration. We were able to use this to convert our cell based readings to an equivalent fluorescein concentration.

Cell Measurement

Transformation

We transformed the plasmids (listed below) resuspended from the Distribution Kit into E. coli DH5α cells. DH5α competent cells were bought from Transgen Biotech. The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates.

Discussion

We did our experiments twice. The first attempt was not so successful as the bacteria grew faster than we assumed. We were not able to keep them at a target OD. Our laboratory is on the first floor, while the plate reader is on the sixth floor. During this period, when we read the number from the machine and ran upstairs to do dilution, the OD had already changed a lot. So we recommend using an ice pack to slow down cell growth in order to obtain better data. All the dilutions and transporting may be performed on ice, just as we did in our second attempt. Additionally, we were not so optimistic about the results of serial dilution and CFU counting as the dilution may not be so accurate, which were easy to be affected by complicated reasons. Our data still did ont look so perfect although we made every step quite carefully and did a second try.