Team:HokkaidoU Japan/Protocols/Infection Assay

From 2011.igem.org

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==T3SS RK13 cell Injection Assay==
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=Preparation of T3SS ''E. coli''=
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===Seed RK13 cells===
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# Remove the culture medium and wash 3 times with PBS follwed by trypsinization
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# Suspend RK13 cells with antibiotics free RPMI-10% FCS
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# 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
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==REQUIRED STUFF==
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===Prepare ''E. coli'' culture===
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*要考慮
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# Grow ''E. coli'' in 4 mL of LB with appropriate antibiotics at 37C overnight
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*epidermal cells of onion
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*reagents
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**MgM-MES pH7.2 (See [ here])
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*************protocol pageにつくる
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**MgM-MES pH5.0
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**MgM-MES pH5.0 with 0.4M Mannitol
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**1% cellulase solution
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**0.1% pectriase in 0.4M Mannitol solution (pH7.0)
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**20% L-arabinose solution
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**Tetracyclin
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**Chroramphenicol
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==PROCEDURE==
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===10 hrs before injection===
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#prepararion of bacterial suspension
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# Centrifuge 4 mL of ''E. coli'' culture at 3,500 rpm for 10 min in the round tube.  
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##Single colony isolation of E.coli that will be used. Cultivation in liquid LB (with no antibiotics) for 2 hours.
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# Centrifuge the culture 3,500 rpm for 10 min
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##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.
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# Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0) with appropriate antibiotics and grow for 4 hrs at 37C
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##Over night cultivation at 37C, 200rpm
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# 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
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##centrifugation of culture fluid at 25C, 3000rpm, 10minutes. Remove supernatant.
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# Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
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##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.
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# Centrifuge the culture 5,000 rpm for 2 min and discard the sup
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##centrifugation of culture fluid at 25C, 3000rpm, 10minutes. Remove supernatant.
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# Repeat step 6 and 7 three times
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##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)
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# Measure and adjust the concentration of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06)
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##Resuspending the perette with 2ml of MgM-MES(pH5.0), adding Tetracycline and Chroramphenicol.
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# And appropriate antibiotics into this ''E. coli'' culture
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##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.
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##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)
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##Removing bacterial culture fluid with pipetteman. Observing using fluorescence microscopes.
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=Infection assay using onion cells=
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===Injection===
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# Remove the RPMI on RK13
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# Add 1 mL of ''E. coli'' RPMI(pH 5.0) culture(ΔOD = 0.06)
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# 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.
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=Infection assay using HeLa cells=
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=Detection of injected protein using GSK tag=
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==Protein extraction from infected HeLa cells==
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==SDS-PAGE and Western Blot analysis==
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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.
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Latest revision as of 14:09, 23 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 in 4 mL of LB 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. Centrifuge the culture 3,500 rpm for 10 min
  3. Discard the sup and resuspend with 4 mL of MgM-MES(pH 5.0) with appropriate antibiotics and grow for 4 hrs at 37C
  4. 4 hrs later(1 hr before injection) centrifuge the culture 3,500 rpm for 10 min, and discard the sup
  5. Resuspend the pellet with 1 mL of antibiotics free RPMI-10% FCS+HCl(pH 5.0) and transfer the culture into micro tube
  6. Centrifuge the culture 5,000 rpm for 2 min and discard the sup
  7. Repeat step 6 and 7 three times
  8. Measure and adjust the concentration of E. coli RPMI(pH 5.0) culture(ΔOD = 0.06)
  9. 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.
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