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:
- 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.
- 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).
- 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.
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 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.
- Attempt 2: In this experiment we used the same electro-competent cells prepared in our laboratory for Attempt 1, on the the other hand we increased the amount of DNA from 1 μL to 2 μL. This attempt also failed because no colonies grew, so we concluded that the unfavorable results related to the Attempt 1 (and also Attempt 2) were not related with the amount of DNA but with the cells.
- Attempt 3: To ensure the best result we decided to use high efficiency competent cells, so in this experiment we used chemo-competent cells DH5- provided by NEB (NEB #C2987I), and we followed the transformation protocol recomended by iGEM except for the amount of DNA, we used the same amount used on Attempt 2. This attempt was successful, the quantity of colonies for each transformation was enough to continue with the experiments.
In every attempt we made one transformation per Device (Test Device 1-6 plus Negative and Positive Controls), if you want to know more about those, please visit Cell Measurement Protocol.
IMPORTANT NOTE: Due to our results in the attempts, we highly recommend future iGEM teams to follow all the protocols recommended by iGEM Headquarters.
- Cell Measurement: After the transformations, we followed the Cell Measurement Protocol in order to measure Fluorescence and Abs600 of 2 transformed colonies of each Test Device and Control in two times, t=0hr (Hour 0) and t=6hr (Hour 6) for each sample. The measurements were made with the same settings used in the calibrations in order to validate the measures. The results of this measurement can be seen in Table III.
Table III: Average data of Fluorescence and Abs600 for each Test Device and Control in both times, Hour 0 and Hour 6. The data was treated subtracting the blank data (LB+Chlor readings).
- CFU per 0.1 OD600 E. coli cultures: To calibrate OD600 to Colony Forming Unit (CFU) count we picked two colonies from each Control (Positive and Negative) to make overnight cultures, those overnight cultures were diluted to reach 0.1 OD600 of each one, and also we made a triplicate for each sample. Every triplicate was serial diluted and then plated in LB+Chlor agar plates to count colonies of each one (to know more about this procedure please see Protocol: Colony Foming Units per 0.1 OD600 E. coli cultures. The results of this experiment can be seen in Table IV.
Table IV: CFU Counting data and mean CFU/mL of each triplicate of each culture in 0.1 OD600. (P1-3, triplicates of first colony picked from Positive Control; P4-6, triplicates of second colony picked from Positive Control; N1-3, triplicates of first colony picked from Negative Control; N4-6, triplicates of second colony picked from Negative Control; D3, dilution 3, with a dilution factor of 8E+4; D4, dilution 4, with a dilution factor of 8E+5; and D5, dilution 5 with a dilution factor of 8E+6).