Team:BostonU/Interlab
Interlab Study
Our team participated in the 2015 iGEM Interlab Study. The interlab study is coordinated effort by iGEM to
gather fluorescence data from three known genetic devices across different labs from around the world.
This is an effort to normalize fluorescence data that may have been recorded under different conditions.
Our team used biobrick cloning methods to make the constructs each of which consisted of a GFP gene
under a different constitutive promoter. The data was collected using FACS. The data was then
processed by negating all fluorescence from cells that were outliers in size or complexity. Then, all cells
that expressed a negative fluorescence value were disregarded.
Procedure
- Inoculate colonies in 500 microliters LB broth overnight
- Inoculate 20 microliters of the overnight culture into 1 mL minimal media with glucode and incubate at 37 degrees Celsius and 300 rpm.
- Check the optical density of 50 microliters culture with 150 microliters LB
- When optical density is between 0.2-0.6 spin down and re-suspend in minimal media and chloramphenicol
- Dilute 40 microliters of resuspended cells into 160 microliter solution of 1.25% paraformaldehyde
- After 30 minutes, dilute 20 microliters of the 1.25% paraformaldehyde solutions into 180 microliters phosphate-buffered saline
- Perform flow cytometry on sample