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iGEM 2011 Team of Hokkaido University

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• Abstract
• What`s T3SS
  Detailed information about T3SS and summary of our achievements on iGEM 2010
• Injection assay using onion cells
  Experiments using plant cells are easier to perform than with mammalian ones
• Ready-to-inject backbone and Bsa I cloning site
  Ready-to-inject backbone and Bsa I cloning site enables easy fusion of T3S signal and protein
• GSK tag system
  A neat injection assay using GSK tag, which can specifically detect successfully injected proteins
• Bsa I cloning site, RFC submission
  Detailed documentation of costructing a BioBrick cloning site a BioBrick!

Injection assay using onion cells

SUMMARY

Last year, we have achieved to inject GFP to the RK13 cells (cultivated rabbit kidney cell) using E. coli which has T3SS. However, using animal cells is not so convenient. For example, we must start to cultivate the cells a couple of days before infection, or deterioration of cultivation environment results in death of cells immediately. Then, we thought up an idea that we might solve these problems by using onion cells. Onion has very wide availability and onion cells is tolerant of bacteria. Moreover, since onion forms detachable monolayer cell-sheets, we thought that we can observe them easily. Thus, we tried repeatedly to establish injection protocol for onion cells. Finally, we were uccessful.

MATERIALS

• E. coli strain we have established last year which can inject GFP into eukaryotic cells.
• Epidermal cell sheet of onion
• Reagents

MgM-MES pH7.2

MgM-MES pH5.0

0.4 M Mannitol in MgM-MES pH5.0

1% cellulase 0.1% pectriase in 0.4M Mannitol solution (pH7.0)

20% L-arabinose solution

Tetracyclin

Chroramphenicol

PROCEDURE

Prepararion of bacterial suspension

1. Isolate a single colony of E. coli and cultured cells in liquid LB (with no antibiotics) for 2 hours.
2. Add 500 ul of culture fluid to 2 ml of liquid LB. Add appropriate amount of antibiotics (Tetracycline and Chloramphenicol). Add 50ul of 20% L-arabinose solution.
3. Culture cells overnight at 37C, 200rpm
4. Centrifuge the cultured cells at 25C, 3000rpm, for 10 min. Remove supernatant.
5. Add MgM-MES (pH5.0), Tetracycline, Chloramphenicol, L-arabinose to the bacterial pellet. Resuspend it using vortex mixer. Culture cells at 37C, 200rpm, 4hours. This step is required because genes encoding salmonella T3SS expresses in acidic environment.
6. Centrifuge the culture fluid at 25C, 3000rpm, for 10 min. Remove supernatant.
7. Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, then centrifuge at 25C, 300rpm, for 10 min. Remove supernatant. Repeat this step for 3 times. (Remove the toxic substances that is produced by E. coli and adjust the osmotic pressure to that in onion cells)
8. Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, Tetracycline, chloramphenicol and L-arabinose.
9. Measure the absorbance at 600 nm using a spectrophotometer, then adjust the concentration of cells to ΔOD = 0.06 by diluting with the same medium mentioned above.
10. For infection of onion cells by E. coli. Adding 500 ul of the culture fluid onto pretreated onion cell-sheets. Leave at RT in petri dishes (Preventing from drying)
11. Remove the bacterial culture fluid carefully by micropipette so that the cell-sheets are not to be torn. Observe the cells under a fluorescence microscope.

Preparation of onion cells

Since we thought that the cell wall may prevent injection of protein, onion cell-sheets were pretreated to remove cell wall as follows.

1. Cut a onion into four pieces
2. Take out a fresh sheets of epidermal cells (second or third layer from the surface of onion).
3. Cut the layer into rectangular (about 1.5cm x 3cm)
4. Tear off a cell sheet from the inner surfase of the layer, stick the sheet onto a slide glass at back face up because the upper face push away enzyme solution.
5. Fix the edge of the sheet onto slide glass with 1% agarose gel.
6. Fill a petri dish with 1% cellulase and 0.1% pectriase in 0.4 M Mannitol solution (pH7.0), then put the cell sheet fixed on slide glass into the enzyme solution.
7. Incubate for 8 min at RT.
8. Prepare 3 petri dishes, filled with MgM-MES pH5.0 containing 0.4 M Mannitol. After processing with enzyme, wash cells stepwise soaking in these petri dishes to remove enzyme solution.
9. Remove excess moisture, and transfer the sample to another petri dishs for infection.

Results

We carried out infection assay using following E. coli:

1. T3SS signal(+)-GFP_T3SS signal(-)-RFP
2. T3SS signal(-)-GFP
3. T3SS only

Here we show the results of infection assey figure 1 to 7. Figure 1 to 3 shows the results of infection assay using the E. coli which we have made last year. That E. coli has T3SS, GFP with secretion signal, and RFP.

Fig. 1 Onion cells after injection was taken under bright field.

Fig. 2 Subcellular localization of GFP after injection. The picture of onion cells was taken under blue light to observe GFP that is fused to secretion signal.

Fig. 3 Localization of RFP in E. coli remaining. The picture was taken under green light to observe RFP that is not fused to secretion signal.

Fig. 4 Onion cells after injection was taken under bright field.

Fig. 5 Localization of GFP signal associated with E. coli cells. The picture of onion cells was taken under blue light to observe GFP that is fused to secretion signal.

GFP not fused to secretion signal is not injected to the onion cells.

Fig. 6 The picture of onion cells after injection was taken under bright field.

Fig. 7 There is no signal under blue light.

We found that utilization of cellulose-treated epidermal cell sheet from onion is useful for quick evaluation of basic ability of E. coli expressing T3SS as protein injector.