Team:US AFRL CarrollHS/InterLab
This year, we completed the Interlab along with the Flow Cytometry portion to qualify for the bronze medal. Below you can find our data including the results of our competent cell test kit, background fluorescence information from out samples, and raw data. This year we were also able to utilize our Flow Cytometer as well to submit our flow cytometry data to iGEM. We hope that by submitting our data to iGEM, we can help standardize measuring protocols for reproducibility within experiments.
In this year’s InterLab, our team transformed E. coli DH5α with different plasmids expressing Green Fluorescent Protein (GFP). A plate reader was then used to measure fluorescence and absorbance values from the transformed strains.
Competent Cells Test
We performed the competent cells test and submitted the data to iGEM.
Figure 1. Plates from the competent cell test
Results
Calibration 1: OD600 Reference point with LUDOX Protocol
Table 1. OD600/Abs600 measurements, showing a correction factor of 2.895
Calibration 2: Particle Standard Curve with Microsphere Protocol
After following the Microsphere Protocol, we were able to produce a Particle Standard Curves in order to observe the relationship between Abs600 and amount of particles present.
Figure 2. Particle Standard Curve
Figure 3. Particle Standard Curve (log scale)
From our data, we observed a linear relationship between Abs600 and particle count.
Table 2. Tabularized Data of Our Results
Calibration 3: Fluorescence standard curve with Fluorescence Protocol
Next we were able to utilize the Microsphere Protocol in order to obtain a standard curve for our fluorescence values.
Figure 4. Fluorescence Standard Curve
Figure 5. FluorescenceStandard Curve (log scale)
Table 3. Tabularized data of Fluorescence Results
Cell Measurement Protocol
Day One
All cells were transformed into E. coli DH5a based on well locations in kit provided by iGEM.
Day Two
Colonies from the transformation plates were then picked and inoculated in 10.0 mL LB broth + Chloramphenicol resistance. The cells were then incubated overnight at 37°C and shaking at 220 rpm.
Day Three
Followed all protocols provided by iGEM, with extra care during serial dilution steps.
Table 4. Raw Plate Reader Measurements
Table 5. Fluorescence Per OD
Table 6. Fluorescence Per Particle