Team:UMaryland/InterLab
InterLab
Synthetic biology is all about collaboration between scientists in an attempt to further our understanding of the field and improve the world around us. However, with the variability that occurs in synthetic biology measurement, it is much harder to consistently build on work done by others. This year, the 5th Interlab Study was designed to reduce lab to lab variability in fluorescence by normalizing data to Colony Forming Units (CFU). The 2018 UMaryland iGEM team has performed the interlab study using the protocol distributed by the Measurement Committee.
Feel free to download our data here.
Feel free to download our data here.
Procedures
All measurements were performed using a standard clear 96-well plate. Calibration of our microplate reader used LUDOX CL-X as a single point reference to appropriately convert absorbance values into a standard OD600 measurement. Absorbances were measured for four replicates per LUDOX and deionized water at 600 nm. The particle standard curve was produced using silica beads and measured at 600 nm. There was also a fluorescence standard curve produced by using fluorescein and 1xPBS solution. Once all calibrations were completed, transformations of all devices in DH5-α, including positive and negative controls took place. Two colonies were chosen from each and incubated in 5 mL of LB media with chloramphenicol. They were left overnight for 16 hours at 37 C at around 220 rpm. All samples were immediately placed on ice and 100 ul was pipetted into the 96 well plate at the 0 and 6h timepoints. The absorbance and fluorescence was measured for both. The CFU measurements were taken by diluting the overnight cultures to an OD of 0.1. The samples were then diluted 1:20 three times and then 1:10 another two times. The final three dilutions for each replicate were plated and colonies were counted after 18 hours of growth at 37 C to determine the CFU/ml as can be seen in our results section.
1. Calibration
OD600 Reference Point - LUDOX Protocol
Particle Standard Curve - Microsphere Protocol
Fluorescence Standard Curve - Fluorescein Protocol
2. Cell Measurement
Plate Reader Raw Measurement
Fluorescence Raw Readings
Hour 0:
Hour 6:
Abs600 Raw Readings
Hour 0:
Hour 6:
Fluorescence per OD
µM Flourescin/OD
Hour 0:
Hour 6:
Net Fluorescin a.u.
Hour 0:
Hour 6:
Hour 0:
Hour 6:
Fluorescence per Particle
MEFL/Particle
Hour 0:
Hour 6:
Fluorescence Raw Readings
Hour 0:
Abs600 Raw Readings
Hour 0:
Fluorescence per OD
µM Flourescin/OD
Hour 0:
Net Fluorescin a.u.
Hour 0:
Fluorescence per Particle
MEFL/Particle
Hour 0:
3. CFUs per 0.1 OD600 E. coli Cultures
Colonies Per Plate
Positive Control
Negative Control
Colony Forming Units per mL
Positive Control
Negative Control
Positive Control
Colony Forming Units per mL
Positive Control
Discussion
Our results indicate an increase in absorbance and fluorescence from 0 hours to 6 hours which indicates that there were more cells present after 6 hours at growth which contributed to a higher fluorescence. In particular, Test Device 1 had the highest fluorescence at both time points. Our positive and negative controls worked as intended and there was very little fluorescence detected from the negative control despite high absorbance indicating the presence of many cells. On average, there was a slight decrease in average fluorescence per molecule and average fluorescence per OD when comparing the 0 hour time point to the 6 hour time point, but it was mainly consistent. The individual cells did not become more or less fluorescent based on cellular concentration in the media.
Contact Us
umarylandigem@gmail.com
Biology - Psychology Building
4094 Campus Dr, College Park, MD 20742
umarylandigem@gmail.com
Biology - Psychology Building
4094 Campus Dr, College Park, MD 20742
© University of Maryland 2018
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