Team:British Columbia/Interlab
INTERLAB
OVERVIEW
The purpose of the 2018 iGEM Interlab study is to establish a standardized GFP measurement protocol that can reduce the variability between different labs performing the same experiments. It is known that when we take measurements with a plate reader to measure OD600, there can be large variability in these measurements due to the difference in the numbers of cells in the sample. This year, the proposed question was, “Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?” The official protocol for the Interlab study can be found on the official iGEM Interlab Study page here. Our data for the Interlab Study is as follows:
METHODS
CALIBRATION
[INSERT GRAPHS HERE]
TRANSFORMATIONS
The eight plasmids (Negative Control, Positive Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, Test Device 6) were transformed into competent E. coli DH5a cells using the E. coli Chemical Transformation protocol here (hyperlink needed to protocol on our wiki). The transformants were then plated on LB + 25 mg/mL Chloramphenicol plates and incubated overnight at 37 degrees Celsius. Two colonies were picked from each plate and inoculated in 5 mL of LB + 25 mg/mL Chloramphenicol overnight at 37 degrees Celsius and 220 rpm.
MEASUREMENTS
[INSERT GRAPHS HERE]
TRANSFORMATIONS
The eight plasmids (Negative Control, Positive Control, Test Device 1, Test Device 2, Test Device 3, Test Device 4, Test Device 5, Test Device 6) were transformed into competent E. coli DH5a cells using the E. coli Chemical Transformation protocol here (hyperlink needed to protocol on our wiki). The transformants were then plated on LB + 25 mg/mL Chloramphenicol plates and incubated overnight at 37 degrees Celsius. Two colonies were picked from each plate and inoculated in 5 mL of LB + 25 mg/mL Chloramphenicol overnight at 37 degrees Celsius and 220 rpm.
======================================================= Cell Measurement: Colony Forming Units: We first measured the OD600 of the positive and negative control inoculations the day after inoculation and diluted the overnight cultures to OD600 = 0.1 in 1 mL of LB + Cam media in triplicate. We then performed serial dilutions according to the protocol and plated them on LB + Cam plates. After 18-20 hours of growth at 37 degrees Celsius, we counted the colonies. (include colony counts here in a table) Results: The detailed results of our Interlab Study can be found here.