InterLab

Calibrations

Three calibrations were performed prior to have standard and more reliable results of Optical Density and Fluorescence in the Cell Measurement. The calibrations performed are next:

1. LUDOX: We used LUDOX CL-X (45% colloidal silica suspension) to transform the absorbance (Abs600) to a corrected value of optical density (OD600) taking in consideration the specifications of the plate. The results of this calibration can be seen in Tables I and II. Table I: Abs600 raw values obtained in the measurements. Table II: Treated values based on the data in Table I.
2. Microsphere Beads: We used monodisperse silica microspheres solution to measure Abs600, the principal idea of this calibration is to standardize a calibration curve of microspheres concentration to be used to convert Abs600 measurements (see Cell Measurements) to an estimated number of cells. We used microsphere beads because those have a similar size and optical characteristics of the cells used in the Cell Measurements. The results of this calibration can be seen in Figure 1.
   
   Figure 1: Particle Standard curves with their respective linear equations. (See normal scale left and log scale right).
3. Fluorescein: We used Fluorescein to make a fluorescence curve, fortunately Fluorescein have similar excitation and emission properties than GFP (the molecule that we measured in the Cell Measurement), and is perfect to make a cost-effective calibration curve of fluorescence to know the amount of GFP in the cells we measured. The results of this calibration can be seen in Figure 2.
   
   Figure 2: Fluorescein Standard curves with their respective linear equations. (See normal scale left and log scale right). Once done the calibrations we moved to the next steps in the Interlab measurement.

Cell measurement

Transformations: Due to execute the best Cell Measurement, we performed different attempts using different transformation protocols in order to compare those and then use the best protocol.

Attempt 1: In this experiment we used electro-competent cells prepared in our laboratory, on the other hand we resuspendended and used the amounts specified in http://parts.igem.org/Help:Protocols/Transformation → the transformation protocol recomended by iGEM. This attempt failed because just a few colonies grew, according to the analysis of the controls used and the quantity of colonies that grew we decided to increase the amount of DNA in the next attempts.