Team:HokkaidoU Japan/Project/Onion

From 2011.igem.org

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==SUMMARY==
==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.
+
Last year, we have achieved injection of GFP to the RK13 cells (cultivated rabbit kidney cell) using ''E. coli'' expressing T3SS. However, using animal cells is inconvenient. For example, cultivation of the cells a must be started couple of days before infection. And deterioration of cultivation environment results in immediate death of cells. We thought that we might solve these problems by using onion cells. Onion has very widely available and its cells are tolerant of bacteria. Moreover, since onion forms detachable monolayer cell-sheets,they can be observe with ease. We repeatedly tried to establish injection protocol for onion cells and after some trials we were successful.
-
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 bactelia. Moreover, since onion forms detatchable 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 successful.
+
==MATERIALS==
==MATERIALS==
-
*the ''E. coli'' strain we have established last year which can inject GFP into eukaryotic cells.
+
* ''E. coli'' strain which can inject GFP into eukaryotic cells that we found last year.  
-
*epidermal cell sheet of onion
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* Epidermal cell sheet of onion  
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*reagents
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* Reagents
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**MgM-MES pH7.2
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:: MgM-MES pH7.2  
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**MgM-MES pH5.0
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:: MgM-MES pH5.0  
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**0.4M Mannitol in MgM-MES pH5.0
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:: 0.4 M Mannitol in MgM-MES pH5.0  
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**1% cellulase 0.1% pectriase in 0.4M Mannitol solution (pH7.0)
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:: 1% cellulase 0.1% pectriase in 0.4M Mannitol solution (pH7.0)  
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**20% L-arabinose solution
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:: 20% L-arabinose solution  
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**Tetracyclin
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:: Tetracyclin  
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**Chroramphenicol
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:: Chroramphenicol  
==PROCEDURE==
==PROCEDURE==
===Prepararion of bacterial suspension===
===Prepararion of bacterial suspension===
-
# Isolate a single colony of ''E. coli'' and cultured cells in liquid LB (with no antibiotics) for 2 hours.
+
# Isolate a single colony of ''E. coli'' and cultured cells in liquid LB (without antibiotics) for 2 hours.  
-
# Add 500 ul of culture fluid to 2 ml of liquid LB. Add proper amount of antibiotics (Tetracycline and Chroramphenicol). Add 50ul of 20% L-arabinose solution.
+
# Add 500 ul of culture fluid to 2 ml of liquid LB. Add appropriate amount of antibiotics (Tetracycline and Chloramphenicol).  
-
# Cultivate over night at 37C, 200rpm
+
# Add 50ul of 20% L-arabinose solution.  
-
# Centrifuge the cultured cells at 25C, 3000rpm, for 10minutes. Remove supernatant.
+
# Culture cells overnight at 37C, shaking at 200rpm  
-
# Add MgM-MES(pH5.0), Tetracycline, Chroramphenicol, L-arabinose to the bacterial perette. Resuspende with vortex. Cultivate at 37C, 200rpm, 4hours. This step is required because T3SS encoded in Salmonella SPI-2 region expresses in acidic environment.
+
# Spin down the cultured cells at 3000rpm, for 10 min at 35C. Remove supernatant.  
-
# Centrifuge the culture fluid at 25C, 3000rpm, 10minutes. Remove supernatant.
+
# Add MgM-MES (pH5.0), Tetracycline, Chloramphenicol, L-arabinose to the bacterial pellet. Resuspend it using vortex mixer. Culture cells at 37C, shaking at 200rpm for 4 hours. This step is required because genes encoding salmonella T3SS are expressed in acidic environment.  
-
# Resuspend the perette with 2ml of MgM-MES(pH5.0) containing 0.4M Mannitol, then centrifuge at 25C, 300rpm, 10 minutes. Remove supernatant. Repeat this step for 3 times. (Remove the toxic substances that is produced by ''E. coli'' and adjust the osmotic pressure to onion cells)
+
# Spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant.  
-
# Resuspende the perette with 2ml of MgM-MES(pH5.0) with 0.4M Mannitol, add Tetracycline, Chroramphenicol and L-arabinose.
+
# Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, then spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant. Repeat this step 3 times. (This is done to remove the toxic substances that are produced by ''E. coli'' and adjust the osmotic pressure to that in onion cells)  
-
# Measur the absorbance 600nm wavelength with a spectrophotometer, then adjust the concentration of culture fluid to delta-OD = 0.06 by diluting with the same medium mentioned above.
+
# Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, Tetracycline, chloramphenicol and L-arabinose.  
-
# Make the onion infected with the ''E. coli'' by adding 500ul of culture fluid onto the processed onion cell-sheets. Leave at RT in petri dishes (Preventing from drying)
+
# Measure the light absorbance at 600 nm using a spectrophotometer, then adjust the concentration of cells to ΔOD600 = 0.06 by diluting with the same medium mentioned above.  
-
# Remove the bacterial culture fluid carefully with micropipette so that the cell-sheets are not to be torn. Observe the cells using a fluorescence microscope.
+
# To infect onion cells with ''E. coli''add 500 ul of the culture fluid onto pre-treated onion cell-sheets. Leave at RT in petri dish (Prevent drying)  
 +
# Remove the bacterial culture fluid carefully by micropipette so that the cell-sheets are not to be torn. Observe the cells under a fluorescencent microscope.  
 +
 
===Preparation of onion cells===
===Preparation of onion cells===
-
We thought that the cell wall might interfere injection of protein, so onion cell-sheets were treated in the following way to dissolve the cell wall.
+
[[File:HokkaidoU Onion Procedure.png|100px|right|]]
-
 
+
Since we thought that the cell wall may prevent injection of protein, onion cell-sheets were pretreated to remove cell wall as follows.  
-
# Cut a onion into four pieces, take out a fresh layer of cells (second or third layer from the surface).
+
# Cut an onion into four pieces.
-
# Cut the layer into rectangular (about 1.5cm x 3cm)
+
# Take out a fresh sheets of epidermal cells (second or third layer from the surface of onion).  
-
# Tear off a cell sheet from the inner surfase of the layer, stick the sheet onto a slide glass at reverse. It is because the outside of the cell sheet sheds enzyme solution.
+
# Cut the layer into rectangular shape (about 1.5cm x 3cm)  
-
# Fasten the sheet onto slide glass by tracing its edge with 1% Agarose gel.
+
# Tear off a cell sheet from the inner surface of the layer.
-
# Fill a petri dish with 1% cellulase and 0.1% pectriase in 0.4M Mannitol solution (pH7.0), then put the slide glass into the enzyme solution.
+
# Put the sheet onto a slide glass at back face up because the upper face deflects enzyme solution, and fix the edge of the sheet onto slide glass with 1% agarose gel.  
-
# Wait for 8min at RT
+
# Fill a petri dish with 1% cellulase and 0.1% pectolyase in 0.4 M Mannitol solution (pH7.0), then submerge the cell sheet fixed on slide glass into the enzyme solution. Incubate for 8 min at RT.
-
# Prepare 3 petri dishes, filled with MgM-MES pH5.0 with 0.4M Mannitol. After processing with enzyme, wash 3 times in these petri dishes to remove enzyme solution.
+
# Prepare 3 petri dishes filled with MgM-MES pH5.0 containing 0.4 M Mannitol. After processing, wash cells stepwise soaking in these petri dishes to remove enzyme solution.  
# Remove excess moisture, and transfer the sample to another petri dishs for infection.
# Remove excess moisture, and transfer the sample to another petri dishs for infection.
-
 
+
<div style="clear:both;"></div>
==Results==
==Results==
-
We carried out infection assay using following ''E. coli'':
+
We carried out infection assay using following ''E. coli'':  
# T3SS signal(+)-GFP_T3SS signal(-)-RFP
# T3SS signal(+)-GFP_T3SS signal(-)-RFP
# T3SS signal(-)-GFP
# T3SS signal(-)-GFP
# T3SS only
# T3SS only
-
Here we show the results of infection assey figure 1 to 7.
+
Here are the results of infection assay 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.
-
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.
+
-
[[File:HokkaidoU_onion_1.JPG|thumb|300px|left|Fig. 1 The picture of the onion cells after infection which was taken under bright field.]]
+
[[File:HokkaidoU_onion_1.JPG|thumb|300px|left|Fig. 1 Onion cells after injection was taken under bright field.]]
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[[File:HokkaidoU_onion_2.JPG|thumb|300px|left|Fig. 2 The same onion cells which was taken under blue laser to observe the GFP.]]
+
[[File:HokkaidoU_onion_2.JPG|thumb|300px|left|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.]]
-
[[File:HokkaidoU_onion_3.JPG|thumb|300px|left|Fig. 3 The same onion cells which was taken under green laser to observe the RFP.]]<div style="clear:both;"></div>
+
[[File:HokkaidoU_onion_3.JPG|thumb|300px|left|Fig. 3 RFP  remains in ''E. coli'' . The picture was taken under green light to observe RFP that is not fused to secretion signal.]]
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In Figure 2, we can see that the onion cells exhibit green fluorescence which came from the injection of GFP. On the other hand, we cannot see that the onion cells exhibit red fluorescence. It is because RFP was not injected because of the lack of the secretion signal.
+
[[File:HokkaidoU_onion_4.JPG|thumb|300px|left|Fig. 4 Onion cells after injection was taken under bright field.]]
 +
[[File:HokkaidoU_onion_5b.JPG|thumb|300px|left|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.]]<div style="clear:both;"></div>
 +
As shown in following pictures GFP not fused to secretion signal is not injected to the onion cells.
-
Figure 4 and 5 shows the results of infection assey using the ''E. coli'' which has T3SS and GFP without T3SS signal.
+
[[File:HokkaidoU_onion_6.JPG|thumb|300px|left|Fig. 6 The picture of onion cells after injection was taken under bright field.]]
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[[File:HokkaidoU_onion_4.JPG|thumb|300px|left|Fig. 4 The picture of the onion cells after infection which was taken under bright field.]]
+
[[File:HokkaidoU_onion_7.JPG|thumb|300px|left|Fig. 7 There is no signal under blue light.]]
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[[File:HokkaidoU_onion_5b.JPG|thumb|300px|left|Fig. 5 The same onion cells which was taken under blue laser to observe the GFP. ]]<div style="clear:both;"></div>
+
-
We cannnot see the green fluorescence inside these onion cells like figure 2. This GFP does not have secretion signal, it is not injected  to the onion cells.
+
-
 
+
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Figure 6 and 7 shows the results of infection assey using the ''E. coli'' which has T3SS but does not have any fluorescence protain.
+
-
[[File:HokkaidoU_onion_6.JPG|thumb|300px|left|Fig. 6 the photo of the onion cells after infection which was taken under bright field.]]
+
-
[[File:HokkaidoU_onion_7.JPG|thumb|300px|left|Fig. 7 There is no signal under blue laser.]]
+
<div style="clear:both;"></div>
<div style="clear:both;"></div>
-
We think the reason of localization of the green fluorescence in Figure 2 comes from the existence of huge vacuole. T3SS cannot pierce a cell membrane and vacuole membrane at once considered from its function[[Team:HokkaidoU_Japan/Project/T3SS#How_does_it_function.3F|*]].  Thus GFP can not enter into a vacuole. Closely observe the onion cells in figure 2, we can see the brightest fluorescence at the cytoplasm, and we can see the weak fluorescence along the contour of the onion cells. It suggest that GFP was injected between cell membrane and vacuole membrane. Although this GFP also has nuclear localization signal, we could not judge whether the GFP localizes in the nucleus or not. But it could be said that the injection assay using onion cells is a valid and easy-to-use method.
+
We found that use of cellulose-treated epidermal cell sheet from onion is useful for quick evaluation of basic ability of ''E. coli'' expressing T3SS as protein injector.  
-
 
+
</div>
</div>
{{Team:HokkaidoU_Japan/footer}}
{{Team:HokkaidoU_Japan/footer}}

Latest revision as of 11:43, 15 December 2011

Contents

  • 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 injection of GFP to the RK13 cells (cultivated rabbit kidney cell) using E. coli expressing T3SS. However, using animal cells is inconvenient. For example, cultivation of the cells a must be started couple of days before infection. And deterioration of cultivation environment results in immediate death of cells. We thought that we might solve these problems by using onion cells. Onion has very widely available and its cells are tolerant of bacteria. Moreover, since onion forms detachable monolayer cell-sheets,they can be observe with ease. We repeatedly tried to establish injection protocol for onion cells and after some trials we were successful.


MATERIALS

  • E. coli strain which can inject GFP into eukaryotic cells that we found last year.
  • 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 (without 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).
  3. Add 50ul of 20% L-arabinose solution.
  4. Culture cells overnight at 37C, shaking at 200rpm
  5. Spin down the cultured cells at 3000rpm, for 10 min at 35C. Remove supernatant.
  6. Add MgM-MES (pH5.0), Tetracycline, Chloramphenicol, L-arabinose to the bacterial pellet. Resuspend it using vortex mixer. Culture cells at 37C, shaking at 200rpm for 4 hours. This step is required because genes encoding salmonella T3SS are expressed in acidic environment.
  7. Spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant.
  8. Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, then spin down the culture fluid at 3000rpm, for 10 min at 25C. Remove supernatant. Repeat this step 3 times. (This is done to remove the toxic substances that are produced by E. coli and adjust the osmotic pressure to that in onion cells)
  9. Resuspend the pellet with 2 ml of MgM-MES (pH5.0) containing 0.4 M Mannitol, Tetracycline, chloramphenicol and L-arabinose.
  10. Measure the light absorbance at 600 nm using a spectrophotometer, then adjust the concentration of cells to ΔOD600 = 0.06 by diluting with the same medium mentioned above.
  11. To infect onion cells with E. coliadd 500 ul of the culture fluid onto pre-treated onion cell-sheets. Leave at RT in petri dish (Prevent drying)
  12. Remove the bacterial culture fluid carefully by micropipette so that the cell-sheets are not to be torn. Observe the cells under a fluorescencent microscope.


Preparation of onion cells

HokkaidoU Onion Procedure.png

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 an 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 shape (about 1.5cm x 3cm)
  4. Tear off a cell sheet from the inner surface of the layer.
  5. Put the sheet onto a slide glass at back face up because the upper face deflects enzyme solution, and fix the edge of the sheet onto slide glass with 1% agarose gel.
  6. Fill a petri dish with 1% cellulase and 0.1% pectolyase in 0.4 M Mannitol solution (pH7.0), then submerge the cell sheet fixed on slide glass into the enzyme solution. Incubate for 8 min at RT.
  7. Prepare 3 petri dishes filled with MgM-MES pH5.0 containing 0.4 M Mannitol. After processing, wash cells stepwise soaking in these petri dishes to remove enzyme solution.
  8. 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 are the results of infection assay 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 RFP remains in E. coli . 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.

As shown in following pictures 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 use of cellulose-treated epidermal cell sheet from onion is useful for quick evaluation of basic ability of E. coli expressing T3SS as protein injector.

Retrieved from "http://2011.igem.org/Team:HokkaidoU_Japan/Project/Onion"