Chiba/Engeneering Flagella

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(Results&Discussion)
(Results&Discussion)
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====Results&Discussion====
====Results&Discussion====
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'''1.Stickiness check using FliC strain'''
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'''1.''Stickiness'' check using FliC strain'''
[[Image:Chiba Beads-Adsorption result3.png|frame|left|fig2. Strain JW1908(''⊿fliC'',).]]<br clear="all">
[[Image:Chiba Beads-Adsorption result3.png|frame|left|fig2. Strain JW1908(''⊿fliC'',).]]<br clear="all">
*Cell without His-FliC bound better to the Beads? No way!
*Cell without His-FliC bound better to the Beads? No way!
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*We thought the problem might be the super-fast revolution of flagella itself. We decided to try MotB strain.<br>
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*We thought the problem might be the super-fast revolution of flagella itself. We decided to try MotB strain.<br><br><br>
[[Image:Chiba_Beads-Adsorption_result2.png|frame|left|fig3. Strain GI826(''⊿fliC'',''⊿motB'').]]<br clear="all">
[[Image:Chiba_Beads-Adsorption_result2.png|frame|left|fig3. Strain GI826(''⊿fliC'',''⊿motB'').]]<br clear="all">

Revision as of 04:46, 27 October 2007

Chiba logo.png

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

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[3].

"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.[4]

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.

Chiba flichisgene.png

  • Chiba check.png Sequence Confirmed
  • Chiba check.png Swarm Confirmed
  • Chiba check.png 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 [5]) 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 using FliC strain

fig2. Strain JW1908(⊿fliC,).

  • Cell without His-FliC bound better to the Beads? No way!
  • We thought the problem might be the super-fast revolution of flagella itself. We decided to try MotB strain.


fig3. Strain GI826(⊿fliC,⊿motB).

  • 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

fig4. Strain JW0747(⊿motB).


    • 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

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