Chiba/Engeneering Flagella

From 2007.igem.org

(Difference between revisions)

Revision as of 21:14, 26 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

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.
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

Kuwajima, G. et al.: Nature Biotechnology, 6, 1080-1083 (1988).
Tanskanen, J. et. al.: Appl. and Env. Microbiol., 4152-4156 (2000)

About Histidine Tag

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

Experiments

Making FliC-his gene

FliC遺伝子のD3領域に、Histidineループをコードする遺伝子を挿入し、ヒスチジンタグとして発現させる。

We inserted the gene of six histidine loop peptide (“His-Tag”).

Sequence Confirmed
Swarm Confirmed
Flagella strained with anti-flagella antibody

Checking the "Stickiness": Beads Adsorption

Purpose

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

Samples

JW1908(⊿fliC株)
- pUC19-fliC-his
- plasmidなし
GI826(⊿fliC⊿motB株)
- pUC19-fliC-his
- plasmidなし

Procedure

pUC19-FliC-His was transformed to JW1908(fliC) and GI826(fliC motB)
培養液をビーズと懸濁させ、大腸菌をBeadsに吸着する。
吸着した大腸菌をbufferで溶出し、incubate。

Results&Discussion

1.Stickiness check

fig2. Strain GI826(fliC,motB).

Plasmidあり、かつCo2+つきビーズ、のコロニー数は、その他のものよりはるかに多い。30倍以上。
Histidine Tagの存在によって大腸菌がビーズに吸着吸着していることがわかる。
The number of colony dramatically decreased with out Co2+ or FliC-His plasmid.

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，FilChisに関してもそれは変わらないみたいな・・・by tashiro

2.Strainの比較（データ？）

Strain	Co²⁺	No metal
JW1908 fliC(-), motB(+)	吸着していない	吸着していない
GI826 fliC(-), motB(-)	吸着していた	吸着していない

@@ Line 21: / Line 21: @@
 ''E.coli'' flagella consist of three parts: a basal body, a hook, and a filament.
-The filament of ''E.Coli'' is a highly rigid, helical, and cylindrical structure which is 10-15μm long and 23nm thick in diameter.
+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 does not take part in the formation of flagella.  (Ref)
+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 possible to display proteins(up to 49.4kDa)on the cell surface of E.Coli using flagellin fusion protein.英語変．
+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]
- 可変なD3ドメインに他の＜__aaまでの＞アミノ酸を挿入しても鞭毛が合成される[2].詳細も書く．
 ====References====

Views

Personal tools