Materials And Methods

BioBricks

Methods

Type III secretion system RK13 cell Injection Assay

Seed RK13 cells

1. Remove the culture medium and wash 3 times PBS followed by trypsinization
2. Suspend RK13 cells with antibiotics free RPMI-10% FCS
3. Seed 6 x 10⁵/well RK13 on the 6 well plate coated with poly-L-Lysin 24 hrs before infection

Prepare E. coli culture

1. Grow E. coli K12 (SPI2/Signal-GFP), E. coli K12 (SPI2) and E. coli K12 (Signal-GFP) in 4 mL of LB (+0.4% Arabinose) with appropriate antibiotics at 37C overnight

5 hrs before injection

1. Centrifuge 4 mL of E. coli culture at 3,500 rpm for 10 min in the round tube
2. Discard the supernatant and resuspend with 4 mL of MgM-MES(pH 5.0) + 0.4% Arabinose with appropriate antibiotics and grow at 37C for 4 hrs
3. 4 hrs later centrifuge the culture 3,500 rpm for 10 min and discard the sup
4. Resuspend the pellet with 2 mL of antibiotics free RPMI-10% FCS and transfer the culture into micro tube
5. Centrifuge the culture 6,000 rpm for 3 min and discard the sup
6. Repeat step 4 and 5 three times
7. Measure and adjust the concentration of E. coli RPMI culture (ΔOD = 0.5)

Injection

1. Remove the RPMI on RK13
2. Add 900 uL of fresh RPMI-10% FCS and then 100 uL of E. coli RPMI culture (ΔOD = 0.5)
3. Incubate the plate at 37C for 1 hrs, 5% CO₂ with or without shaking
4. Wash the wells with penisilin/ strepromicin containing RPMI-10% FCS twice
5. Add 2 mL of penisilin/ streptomicin containing RPMI-10% FCS/well
6. Incubate the plate at 37C for 1 hr, 5% CO₂
7. Observe the cells by fluorescence microscope under blue light at every 30 min