Team:HokkaidoU Japan/Protocols/Infection Assay

From 2011.igem.org

(Difference between revisions)
m
Line 3: Line 3:
<div id="hokkaidou-right-content">
<div id="hokkaidou-right-content">
-
=Preparation of T3SS ''E. coli''=
+
==T3SS RK13 cell Injection Assay==
 +
===Seed RK13 cells===
 +
# Remove the culture medium and wash 3 times with PBS follwed by trypsinization
 +
# Suspend RK13 cells with antibiotics free RPMI-10% FCS
 +
# Seed 2x10<sup>5</sup>/well RK13 cells on a 6 well plate or a glass bottom 3.5 cm dish 20 hrs before infection
 +
===Prepare ''E. coli'' culture===
 +
# Grow E.coli-K12(SPI2/Signal-GFP/RFP), and ''E. coli'' K12(SPI2)in 4 mL of LB+0.4% arabinose with appropriate antibiotics at 37C overnight
-
=Infection assay using onion cells=
+
===10 hrs before injection===
 +
# Centrifuge 4 mL of ''E. coli'' culture at 3,500 rpm for 10 min in the round tube.
 +
# Discard the sup and resuspend with 4 mL of MgM-MES(pH 7.2)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
 +
# 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min
 +
# Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
 +
# 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
 +
# Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
 +
# Centrifuge the culture 5,000 rpm for 2 min and discard the sup
 +
# Repeat step 6 and 7 three times
 +
# Measure and adjust the concentration of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06)
 +
# Add 0.4% arabinose and appropriate antibiotics into this ''E. coli'' culture
-
 
+
===Injection===
-
 
+
# Remove the RPMI on RK13
-
=Infection assay using HeLa cells=
+
# Add 1 mL of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06)
-
 
+
# Incubate the plate at 37C, 5%CO<sub>2</sub> and observe the cells by Cofocal Laser Scannning Microscope(OLYMPUS FV-1000D) under blue and green exciter light at every 1.5 hrs after first exposure.
-
 
+
-
 
+
-
=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.
+
</div>
</div>
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Revision as of 03:35, 6 October 2011

Contents

T3SS RK13 cell Injection Assay

Seed RK13 cells

  1. Remove the culture medium and wash 3 times with PBS follwed by trypsinization
  2. Suspend RK13 cells with antibiotics free RPMI-10% FCS
  3. Seed 2x105/well RK13 cells on a 6 well plate or a glass bottom 3.5 cm dish 20 hrs before infection

Prepare E. coli culture

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

10 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 sup and resuspend with 4 mL of MgM-MES(pH 7.2)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
  3. 4 hrs later(5.5 hrs before injection) centrifuge the culture 3,500 rpm for 10 min
  4. Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0)+0.4% arabinose with appropriate antibiotics and grow for 4 hrs at 37C
  5. 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
  6. Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
  7. Centrifuge the culture 5,000 rpm for 2 min and discard the sup
  8. Repeat step 6 and 7 three times
  9. Measure and adjust the concentration of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
  10. Add 0.4% arabinose and appropriate antibiotics into this E. coli culture

Injection

  1. Remove the RPMI on RK13
  2. Add 1 mL of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
  3. Incubate the plate at 37C, 5%CO2 and observe the cells by Cofocal Laser Scannning Microscope(OLYMPUS FV-1000D) under blue and green exciter light at every 1.5 hrs after first exposure.
Retrieved from "http://2011.igem.org/Team:HokkaidoU_Japan/Protocols/Infection_Assay"