Difference between revisions of "Team:British Columbia/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:

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. 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

CELL MEASUREMENTS

From the previous day’s inoculations, we then made a 1:10 dilution of each overnight culture in LB + Chloramphenicol (0.5 mL of culture into 4.5 mL of LB + Chlor). We then measured the Abs600 of these 1:10 diluted cultures and diluted the cultures further to a target Abs600 of 0.02 into a final volume of 12 ml LB + Chlor in test tubes that were covered in foil.

We took 500 uL samples of the diluted cultures at 0 hours into 1.5 mL eppendorf tubes, prior to incubation, and placed them on ice. We then incubated the remainder of the cultures at 37 degrees Celsius and 220 rpm for 6 hours. After 6 hours, we took 500 uL samples of the cultures and put them into 1.5 mL eppendorf tubes and put them on ice. After each time point, we measured the Abs600 and fluorescence of them according to the plate diagram in the protocol.

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.

RESULTS

The detailed results of our Interlab Study can be found here.