▪ Aug 15
Invasin + Listerolysin
☀ Yi Han ☀
Invasion assay with HEK293T cells and BL21, and BL21+inv-hly plasmid on 15/08/15
Rationale: The invasin gene in the invasin+listerolysin plasmid should remain intact and be expressed, leading to an invasion phenotype
Objective: Screen invasion phenotype of BL21 and BL21+invasin-listerolysin Biobricks part
Experiment details
Cells were passage 2
Seed 0.5mL of 5x10^5 HEK293T cells/mL and 1x10^6 in wells of 24-well plates, grow overnight.
Determine cell confluency with microscopy the next day - 80-90% confluency
All strains were grown overnight in 3mL LB broth
For 5x10^5 cells
100ul of 10^9 cells/mL was used for moi 200
100ul of 2X dilution of 10^9 cells/mL was used for moi 100
100ul of 4X dilution of 10^9 cells/mL was used for moi 50
100ul of 5X dilution of 10^9 cells/mL was used for moi10
Results
Lawn on all plates
HEK293T cells detach too easily. Grow for 24 hours in the future and do minimal and gentle washing.
For next experiment, 0.5x10^6 and 1x10^6 cells wre seeded
1000ug/mL kanamycin for 1 hour is sufficient for kill step
no contamination in uninfected control
There is invasion in background BL21 strain as well
All mois showed high percentage of infection
In future use moi 10:1 and plate up 10^2 to 10^5 dilution for countable colonies
