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	**pUC19-fliC-his		**pUC19-fliC-his
	**no plasmid		**no plasmid
-	*⊿fliC⊿motB strain(GI826)transformed with	+	*⊿fliC,⊿motB strain(GI826)transformed with
	**pUC19-fliC-his		**pUC19-fliC-his
	**no plasmid		**no plasmid
	<br>		<br>
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	====Testing Procedure====		====Testing Procedure====

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Revision as of 04:21, 27 October 2007

Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal Acknowledgements | Team Members | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | 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.

[http://www.npn.jst.go.jp/index.html 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]

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

• ⊿fliC strain(JW1908 in KEIO collections [3]) transformed with
  • pUC19-fliC-his
  • no plasmid
• ⊿fliC,⊿motB strain(GI826)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

• Histidineが発現していないほうがBeadsに吸着している。
• 鞭毛が発現していないほうが吸着度合いが強いことから、鞭毛の働きによって、逆に、吸着力が弱くなってしまっている。
• 鞭毛の回転が問題なのではないか。

• In the presence　of Co²⁺Histidine tag,beads adsorb bacteria.

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

• In the presence of FliC-His, cobalt ion adsorb bacteria stronger than nickel ion.
• 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
  • 鞭毛の回転がなければある程度の吸着力を保つ。しかしワイルドタイプのFliCの発現も伴うため、Histidineによる吸着度合いは低下してしまう。

3.金属イオンの比較 一般的に知られているように、コバルトのほうがHistidineとより結合をつくるのではないか。

References

1. Kuwajima, G. et al.: J. Bacteriol., 170, 3305-3309 (1988)
2. Ezaki, S. et. al.: J. Ferment. Bioeng., 86, 500-503 (1998)
3. Baba, T. et. al.: Mol. Systems. Biol., 21, 1-10 (2006)