Team:Tec-Monterrey/InterLab
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Interlab
Fifth International InterLab Measurement Study
Procedure
Interlab procedure was made to achieve bronze #4 requirement. To make this, the IGEM 2018 Interlab Study Protocol was followed. To make calibration and experimental data the following material was used: measurement kit which contains Ludox, silica bead and fluorescein, competent cells from E. coli strain DH5a and 96 well plates, black with a clear flat bottom. The first part of the protocol was to make standard curves for OD measurements, cell standard curves and fluorescein standard curves. Using Ludox solution, it was capable to obtain the following reference point for an OD600.
The obtain conversion value obtained is 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 realized to obtain a standard curve using silica microspheres, which have the same size and optical characteristics of bacteria cells. The standard curve was used to convert ABS600 to an estimated number of cells. The following standard curve was obtained.
Figure 1: Normal Table with values
Finally, a fluorescein standard curve was made to calculate the fluorescence values of transform 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 was possible to proceed to the cell measurement protocolos with transform E.coli DH5a strains. The following devices from the IGEM kit were transformed.
Figure 1: Normal Table with values
Results
Absorbance
Absorbance of cells at t=0h and t=6 h are shown in the following diagram.
Figure 1: Normal Table with values
It can be seen that for all transform bacteria growth after 6 hours. Some of the devices, such as device 1 and 5 didn´t growth sufficiently as other transform bacteria.
Absorbance
Fluorescences values at t=0h 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/ little fluorescence at both time. Device 3 didn’t show any fluorescence at all which affect the measurements of it. The device that show the greater fluorescence value was device 4.