HokkaidoU Japan

iGEM 2011 Team of Hokkaido University

Preparation of T3SS E. coli

REQUIRED STUFF

要考慮
epidermal cells of onion
reagents
- MgM-MES pH7.2 (See [ here])
  - - - protocol pageにつくる
- MgM-MES pH5.0
- MgM-MES pH5.0 with 0.4M Mannitol
- 1% cellulase solution 0.1% pectriase in 0.4M Mannitol solution (pH7.0)
- 20% L-arabinose solution
- Tetracyclin
- Chroramphenicol

PROCEDURE

prepararion of bacterial suspension
1. Single colony isolation of E.coli that will be used. Cultivation in liquid LB (with no antibiotics) for 2 hours.
2. Adding 500ul of culture fluid to 2ml of liquid LB. Adding proper amount of antibiotics (Tetracycline and Chroramphenicol). Adding 50ul of 20% L-arabinose solution.
3. Over night cultivation at 37C, 200rpm
4. centrifugation of culture fluid at 25C, 3000rpm, 10minutes. Remove supernatant.
5. Adding MgM-MES(pH5.0), Tetracycline, Chroramphenicol, L-arabinose to the bacterial perette. Resuspending with vortex. Cultivation at 37C, 200rpm, 4hours. This step is required because T3SS express in acidic environment.
6. centrifugation of culture fluid at 25C, 3000rpm, 10minutes. Remove supernatant.
7. Resuspendding the perette with 2ml of MgM-MES(pH5.0), then centrifugation at 25C, 300rpm, 10 minutes. Remove supernatant. Repeat this step for 3 times. (Removing the toxic substances which E.coli produce and adjusting the osmotic pressure of onion cells)
8. Resuspending the perette with 2ml of MgM-MES(pH5.0), adding Tetracycline and Chroramphenicol.
9. Measuring the absorbance 600nm wavelength with spectrophotometer, adjusting the concentration of culture fluid to delta-OD = 0.06 by diluting with MgM-MES(pH5.0) with Mannitol. Adding proper amount of Tetracycline, Chroramphenicol, and L-arabinose.
10. Making onion infected with E.coli by adding 500ul of culture fluid to processed onion cell-sheets. Leaving at RT in petri dishes (Preventing from drying)
11. Removing bacterial culture fluid with pipetteman. Observing using fluorescence microscopes.

Infection assay using onion cells

Infection assay using HeLa cells

Detection of injected protein using GSK tag

Protein extraction from infected HeLa cells

SDS-PAGE and Western Blot analysis

HeLa cell lysates were subjected to SDS-PAGE, and separated proteins were transferred to an Immobilon-P membranes (Millipore). The membranes were blocked with Blocking buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature. The blots were probed with Phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology #9336) or GSK-3β antibody (Cell Signaling Technology #9315) diluted 1/1000 in Blocking buffer and incubated overnight at room temperature. Blots were washed three times with TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 15 min each time. Secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) was diluted 1/1000 in Blocking buffer and incubated with the blots for 1.5 h at 37C. Blots were washed as described above and developed with BCIP/NBT.