Team:Tec-Monterrey/InterLab

Procedure

In order to achieve Bronze requirement #4, the Interlab procedure was successfully carried out, following the iGEM 2018 Interlab Study Protocol. To obtain calibration and experimental data, the following materials were used: competent cells from E. coli 's strain DH5α, 96-well plates (black with a clear flat bottom), and the measurement kit, which contains Ludox, silica beads and fluorescein. The first part of the protocol involved generating standard curves for OD measurements, cells and fluorescein. Using a Ludox solution, it was possible to obtain the following reference point for an OD₆₀₀. The conversion value obtained was then used to convert any measurement of cell density into OD₆₀₀ by multiplying by 2.897, using the same instrument under the same conditions.

The particle standard curve was obtained using silica microspheres, which have the same size and optical characteristics as bacteria cells. The standard curve was then used to convert ABS₆₀₀ to an estimated number of cells. The following standard curve was obtained:

Figure 1: Calibration curve of particle count from Absorbance at 600 nm.

Finally, a fluorescein standard curve was made to calculate the fluorescence values of transformed bacteria with GFP. The filter used for the measurements was used according to the recommended values (Bandpass: 530 nm/ 30 nm, width: 25-30 nm, excitation 485 nm and emission 520-530 nm). The curve displayed in Figure 2 was obtained:

Figure 2: Calibration curve of Fluorescein

Using the previous standard curves, it was possible to continue with the cell measurement protocols with transformed E. coli DH5a strains. The following devices from the iGEM kit were transformed:

Figure 4: Devices used to transform DH5α for fluorescence standardising.

Results

Absorbance

Absorbance of cells at t=0 h and t=6 h are shown in the diagram of Figure 5.

Figure 5: Absorbance at 600 nm of transformed bacteria with devices from iGEM kit.

All transformed bacteria show growth after 6 hours. Some of the devices, such as device 1 and 5, did not grow sufficiently as other transformed bacteria did.

Fluorescence

Fluorescence values at t = 0 h and t = 6 h

Figure 6: Fluorescence of transformed bacteria with devices from iGEM kit.

Conclusion

Fluorescence values obtained by GFP show that some devices emit greater fluorescence values than others. The negative device show no to little fluorescence at both times. Device 3 did not show any fluorescence at all, which affected its measurements. The device that shows the greater fluorescence value was device 4.
From absorbance and fluorescence values it is evident that fluorescence is not directly related to the culture's optical density, which is to say that absolute GFP expression is not directly related to the amount of bacteria, as it depends on each device. It is then possible to standardise each of these GFP devices, comparing the results from the iGEM Teams that carried out this InterLab protocol, so that amount of bacteria can be related to fluorescence produced by each device.

Team:Tec-Monterrey/InterLab

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