Interlab

We are willing to participate in iGEM 2018 InterLab Study, thanks to iGEM HQ provided us this optunity to share our results with many iGEMers. For improving reproducibility, we use plate readers to take measurements of fluorescence and absorbance in accordance with the requirements.

Before we begin our experiments, we had known all the informations about our plate reader, which are provided when we submitted our data to iGEM HQ. And we prepared all of the supplies and reagents used in the experiments.

1.Calibrations

For all of the calibration measurements, we used the same plates and volumes, we also used the same settings, such as filters, excitation and emission wavelengths, temperatures, etc.

1.1 Calibration 1: OD600 Reference point

According to the LUDOX Protocol, 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 the plate reader into a comparable OD600 measurement obtained in a spectrophotometer.

NOTE: we did not find an automatic path length correction on our instrument. After finish all the processed, we obtained our correction factor to convert measured Abs600 to OD600, it is 6.222, so all the Abs600 readings using this instrument with the same settings and volume were converted to OD600 by multiplying by 6.222.

1.2 Calibration 2: Particle Standard Curve

We have known that the size and optical properties of the silica microspheres are similar to cells, and there is a known amount of particles per volume. In accordance with the protocol, we prepared a dilution series of monodisperse silica microshperes (4 replicates) and measured the Abs600 in our plate reader. These measurements allowed us to construct a particle standard curve which was used to convert Abs600 measurements to an estimated number of cells.

NOTE: 1.Accurate dilution is important here which depends on the accurate Micropipettes and right using method; 2.Each well should be mixed immediately before putting in the plate reader. 3.Avoiding to create bubbles on the liquid surface is another important factor affecting the measurement. In all the measurement process, we tried our best to finish perfectly.

1.3 Calibration 3: Fluorescence standard curve – Fluorescein Protocol

Since plate readers report fluorescence values in arbitrary units that vary widely from instrument to instrument, absolute fluorescence values cannot be directly compared from one instrument to another. For comparing fluorescence output of test devices between teams, we are required to create a standard fluorescence curve. We used the small molecule fluorescein, which has similar excitation and emission proterties to GFP, but is cost-effective and easy to prepare instead of GFP. We prepared a dilution series of fluorescein in 4 replicates and measured the fluorescence in a 96 well plate, black with clear flat bottom, in our plate reader. By measureing these wells, we generated a standard curve of fluorescence for fluorescein concentration. So we can use this to convert our cell based readings to an equivalent fluorescein concentration.

2.Cell measurement

2.1 Raw plate reader measurements

After we completed the three calibrations, we started the cell measurement under the same conditions: the same plates and volumes, the same settings and same temperature, etc. For the sake of consistency and reproducibility, we used E.coli K-12 DH5-α as competent cells. We prepared all the materials and devices as the protocols, and prepared all the samples as the workflow.

According to the measurement protocols, samples were laid out as the plate diagram below. Pipette 100ul of each sample into each well. From 500ul samples in a 1.5ml eppendorf tube, 4 replicate samples of colony #1 were pipetted into wells in rows A, B, C and D. Replicate samples of colony #2 were pipetted into wells in rows E, F, G and H. Only LB+chloramphenicol on each plate in column 9, as shown in the diagram below.

At the end of the experiment, we had two plates to read. Each plate was set up shown above. We had one plate for each time point: 0 and 6 hours. Ohe each plate we read both fluorescence and absorbance. The data are shown as below.

Team:JNFLS/Interlab