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 material were used: competent cells from E. coli's strain DH5a, 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.

Figure 1: Table for the conversion value of OD₆₀₀.

The particle standard curve was obtained using silica microspheres, which have the same size and optical characteristics of 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: Normal Dsitribution Table with values

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, exicitation 485 nm and emission 520-530 nm). The following curves were obtained:

Figure 1: Normal Table with values

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

Figure 1: Normal Distribution Table with values

Results

Absorbance

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

Figure 1: Normal Table with values

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.

Absorbance

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

Figure 1: Normal Table with values

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.

Team:Tec-Monterrey/InterLab

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