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Revision as of 01:44, 27 October 2007 (view source) Katabami (Talk | contribs) (→References) ← Older edit		Revision as of 01:47, 27 October 2007 (view source) Umeno (Talk | contribs) (→Procedure) Newer edit →
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	**no plasmid		**no plasmid
-	====Procedure====	+	====Testing Procedure====
	#pUC19-FliC-His was transformed to JW1908(''fliC'') and GI826(''fliC'' ''motB'').		#pUC19-FliC-His was transformed to JW1908(''fliC'') and GI826(''fliC'' ''motB'').
-	#Incubated 4 cell samples written above.	+	#Grown to stationary phase
-	#Culture was suspended with beads which will stick to histidine.	+	#Culture suspended with Dynabeads (Metal-IDA), allowing to the affinity adsorption
-	#Adsorbed ''E.coli'' on the beads was washed with a buffer and spreaded on agar plates.	+	#Beads washed with a phosphate buffer (x4)
-	#The colony of each plate was counted.	+	#E" coli" detached from beads by adding imidazole then spreaded on agar plates.
		+	#The number of the colonies on resultant plates.
	====Results&Discussion====		====Results&Discussion====

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Revision as of 01:47, 27 October 2007

Introduction | Project Design ( 1.Sticky Hands | 2.Communication | 3.Size Control ) | Making Marimos | Our Goal || Team Members | メンバ連絡簿

Stickey Tags

Our Aim

To make stickey hands on E.coli, we focused on their flagella that are located outside the cells. We used the following mechanisms:

• Display sticky peptides in flagellar filament.
• His-tag. The imidazole group in histidines make a complex with metal ions.　

We combined these two and made a His-tagged flagella in the hope to stick them together via metal ions.

About flagella

E.Coli have 5-10 flagella. The flagella is used for swimming and for chemotaxis; the bacteria run when they find attractant, tumble when there is a repellent.

E.coli flagella consist of three parts: a basal body, a hook, and a filament. The filament of E.Coli is a rigid, helical, and cylindrical structure which is 10-15μm long and 23nm thick in diameter. It is built from ~20000 subunits of a ~55kDa single protein, FliC. FliC has three domains, D1,D2,D3; although D1 and D2 are needed for the formation of the functional flagellar filament, D3 domain which sticks outside of the fillament are not essential[1].

"Variable" FliC D3 domain

It is reported that the proteins up to 49.4kDa could be displayed on the cell surface of E.Coli using flagellin fusion protein.[2]

References

1. Kuwajima, G. et al.: J. Mol. Bacteriol., 170, 3305-3309 (1988).
2. Ezaki, S. et. al.: J. Ferment. Bioeng., 86, 500-503 (1998)

About Histidine Tag

See [http://en.wikipedia.org/wiki/His-tag wikipedia article].

Experiments

Making FliC-his gene

• We inserted the short peptide with six histidine (“His-Tag”) into the fliC D3 domain.

• Sequence Confirmed
  
• Swarm Confirmed
  
• Flagella strained with anti-flagella antibody

Checking the "Stickiness": Beads Adsorption

Purpose

Confirm that the his-tags are displaied on the flagella and are capable of binding to Co²⁺- or Ni²⁺- surface.

Samples

• JW1908(⊿fliC strain) transformed with
  • pUC19-fliC-his
  • no plasmid
• GI826(⊿fliC⊿motB strain) transformed with
  • pUC19-fliC-his
  • no plasmid

Testing Procedure

1. pUC19-FliC-His was transformed to JW1908(fliC) and GI826(fliC motB).
2. Grown to stationary phase
3. Culture suspended with Dynabeads (Metal-IDA), allowing to the affinity adsorption
4. Beads washed with a phosphate buffer (x4)
5. E" coli" detached from beads by adding imidazole then spreaded on agar plates.
6. The number of the colonies on resultant plates.

Results&Discussion

1.Stickiness check

• In the presence　of Co²⁺Histidine tag,beads

Co²⁺Histidine Tagの存在よって大腸菌がビーズに吸着している．

• Ni²⁺よりもCo²⁺のほうがfliC-his存在下でより吸着している．
• Ni²⁺よりもCo²⁺のほうがfliC-hisの有無で吸着の差が大きい
• The number of colony dramatically decreased with out Co2+ or FliC-His plasmid.

2.Strainの比較

• この実験結果から言えることは何ですか？それと、上の実験結果との比較は？byとよたろ