Chiba/Engeneering Flagella

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Revision as of 03:44, 27 October 2007

Stickey Tags

Our Aim

Fig1.Bacteria Linker

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]

References

Kuwajima, G. et al.: J. Bacteriol., 170, 3305-3309 (1988).
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

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

Results&Discussion

1.Stickiness check

fig2. Strain GI826(⊿fliC,⊿motB).

[[Image:|frame|left|fig2. Strain GI826(⊿fliC,⊿motB).]]

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

fig2. Strain JW0747(⊿motB).

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

3.金属イオンの比較

@@ Line 69: / Line 69: @@
 .Stickiness check
 [[Image:Beads-Adsorption Colony-Number.jpg|frame|left|fig2. Strain GI826(''⊿fliC'',''⊿motB'').]]<br clear="all">
+[[Image:|frame|left|fig2. Strain GI826(''⊿fliC'',''⊿motB'').]]<br clear="all">
 *In the presence　of Co<sup>2+</sup>Histidine tag,beads
 Co<sup>2+</sup>Histidine Tagの存在よって大腸菌がビーズに吸着している．
@@ Line 77: / Line 78: @@
 <!--
 *'''FliC(hisなし)はないんでしたっけ？古'''
-**ないです。今から取るか否か、というところです。結果が出るのはwikiの締め切りの後（明日の昼）です。Wikiにはどのように考察、記載するべきでしょうか？Tominaga
+**ないです。今から取るか否か、というところです。結果が出るのはwikiの締め切りの後（明日の昼）です。Wikiにはどのように考察、記載するべきでしょうか？TominagaBeads-Adsorption Colony-Number.jpg
 *'''考察も書いたら？　なぜコバルトがニッケルより良いのか？一般的にニッケルよりコバルトの方が結合力が強いことが知られてて，FilC-hisに関してもそれは変わらないみたいな・・・by tashiro'''
 **Beadsとヒスチジンの反応では、Co2+が中心に位置し、ヒスチジンの空間的位置に対する要求性が厳密になっています。連続するヒスチジンや空間的に適切に配置する隣接ヒスチジンのみがこの反応中心でコバルトに結合します。Ni2+ではこのような空間的要求性はあまり厳密ではありません。このため、Ni2+つきのBeadsを用いた場合、His タグ融合タンパク質以外に存在するヒスチジンも結合してしまいます。

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