Chiba/Engeneering Flagella

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[[Image:chiba_logo.png|center]]
[[Image:chiba_logo.png|center]]
__NOTOC__
__NOTOC__
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{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
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[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]<br>
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[[Chiba|Home]]<br>
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<span style="font-size:120%;font-weight:bold;">[[Chiba/Introduction|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]</span><br>
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]  
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==Affinity Tags==
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==Stickey Tags==
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===Our Aim===
===Our Aim===
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[[Image:Chiba_stickbacteria.png|frame|'''Fig1.'''Bacteria Linker]]
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[[Image:Chiba_stickbacteria.png|frame|'''Fig. 9''' Affinity tags.]]
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To make stickey hands on ''E.coli'', we focused on their flagella that are located outside the cells. We used the following mechanisms:
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To make affinity tags 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.
*Display sticky peptides in flagellar filament.
*His-tag. The imidazole group in histidines make a complex with metal ions.　
*His-tag. The imidazole group in histidines make a complex with metal ions.　
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The filament of ''E.Coli'' is a 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.
It is built from ~20000 subunits of a ~55kDa single protein, FliC.
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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].
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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====
===="Variable" FliC D3 domain====
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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]
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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]
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===About Histidine Tag===
===About Histidine Tag===
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*We inserted the short peptide with six histidine (“His-Tag”) into the fliC D3 domain.  
*We inserted the short peptide with six histidine (“His-Tag”) into the fliC D3 domain.  
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[[Image:Chiba_flichisgene.png]]<br>
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[[Image:Chiba_flichisgene.png|frame|'''Fig.10''' flic his gene]]<br>
*[[Image:Chiba_check.png]] Sequence Confirmed<br>
*[[Image:Chiba_check.png]] Sequence Confirmed<br>
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====Samples====
====Samples====
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*⊿fliC strain(JW1908 in KEIO collections [3]) transformed with
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*⊿fliC strain(JW1908 in KEIO collections [5]) transformed with
**pUC19-fliC-his
**pUC19-fliC-his
**no plasmid
**no plasmid
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*⊿fliC⊿motB strain(GI826)transformed with
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*⊿fliC,⊿motB strain(GI826)transformed with
**pUC19-fliC-his
**pUC19-fliC-his
**no plasmid
**no plasmid
<br>
<br>
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====References====
 
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3. Baba, T. ''et. al''.: ''Mol. Systems. Biol.,'' '''21''', 1-10 (2006)
 
====Testing Procedure====
====Testing Procedure====
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#pUC19-FliC-His was transformed to JW1908(''fliC'') and GI826(''fliC'' ''motB'').
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#pUC19-FliC-His was transformed to JW1908(''fliC''), JW0747(''MotB''), and GI826(''fliC'' ''motB'').
#Grown to stationary phase
#Grown to stationary phase
#Culture suspended with Dynabeads (Metal-IDA), allowing to the affinity adsorption
#Culture suspended with Dynabeads (Metal-IDA), allowing to the affinity adsorption
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====Results&Discussion====
====Results&Discussion====
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1.Stickiness check
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'''1.''Stickiness'' check using FliC strain'''
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[[Image:Chiba Beads-Adsorption result3.png|frame|left|fig2. Strain JW1908(''⊿fliC'',).]]<br clear="all">
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[[Image:Chiba Beads-Adsorption result3.png|frame|left|'''Fig. 11''' Binding test using Strain JW1908(''⊿fliC'',).]]<br clear="all">
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*Histidineが発現していないほうがBeadsに吸着している。
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*Cell without His-FliC bound better to the Beads? No way!
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*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。
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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>
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*鞭毛の回転が問題なのではないか。
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[[Image:Chiba_Beads-Adsorption_result2.png|frame|left|fig3. Strain GI826(''⊿fliC'',''⊿motB'').]]<br clear="all">
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'''2.''Stickiness'' check using MotB strain'''
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*In the presence　of Co<sup>2+</sup>Histidine tag,beads adsorb bacteria.
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[[Image:Chiba Beads-Adsorption result2.png|frame|left|'''fig. 12''' Binding test using Strain JW0747(''⊿motB'').]]<br clear="all">
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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．
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*Mot B deletion provides cell with the flagella completely assembled but not rotating. <br>
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*In the presence of FliC-His, cobalt ion adsorb bacteria stronger than nickel ion.
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*'''This time everything worked out!''' Only in the presence of Co<sup>2+</sup>Bacteria with His-FliC sticked to the Co-IDA beads very well.
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*Ni<sup>2+</sup>よりもCo<sup>2+</sup>のほうがfliC-his存在下でより吸着している．
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*In this strain, FliC-His is assembled with wildtype FliC coded in genomic DNA. Nevertheless, the binding efficiency was at the same level (not shown). it seems that His-Tag displayed on the flagella is enough to do its work.
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*Ni<sup>2+</sup>よりもCo<sup>2+</sup>のほうがfliC-hisの有無で吸着の差が大きい
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*On the other hand, the deletion of MotB turned out to be vital for sticking the tagged flagella together.
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*The number of colony dramatically decreased with out Co2+ or FliC-His plasmid.
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*In the presence of FliC-His, cobalt ion adsorb bacteria stronger than nickel ion, this was more or less the expected result.
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2.Strainの比較
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==References==
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[[Image:Chiba Beads-Adsorption result2.png|frame|left|fig4. Strain JW0747(''⊿motB'').]]<br clear="all">
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3. Kuwajima, G. ''et al''.: ''J. Bacteriol.'', '''170''', 3305-3309 (1988)<br>
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<br>
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4. Ezaki, S. ''et. al''.: ''J. Ferment. Bioeng.'', '''86''', 500-503 (1998)<br>
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*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
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5. Baba, T. ''et. al''.: ''Mol. Systems. Biol.,'' '''21''', 1-10 (2006)<br>
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**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。
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3.金属イオンの比較
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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。
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====References====
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#Kuwajima, G. ''et al''.: ''J. Bacteriol.'', '''170''', 3305-3309 (1988)
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#Ezaki, S. ''et. al''.: ''J. Ferment. Bioeng.'', '''86''', 500-503 (1998)
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#Baba, T. ''et. al''.: ''Mol. Systems. Biol.,'' '''21''', 1-10 (2006)
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Latest revision as of 05:26, 27 October 2007

Chiba logo.png

Home
Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal
Acknowledgements | Team Members | iGEM Chiba Website | メンバ連絡簿

Affinity Tags

Our Aim

Fig. 9 Affinity tags.

To make affinity tags 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[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 wikipedia article.

Experiments

Making FliC-his gene

  • We inserted the short peptide with six histidine (“His-Tag”) into the fliC D3 domain.
Fig.10 flic his gene

  • 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), JW0747(MotB), 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

Fig. 11 Binding test using 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.


2.Stickiness check using MotB strain

fig. 12 Binding test using Strain JW0747(⊿motB).

  • Mot B deletion provides cell with the flagella completely assembled but not rotating.
  • This time everything worked out! Only in the presence of Co2+Bacteria with His-FliC sticked to the Co-IDA beads very well.
  • In this strain, FliC-His is assembled with wildtype FliC coded in genomic DNA. Nevertheless, the binding efficiency was at the same level (not shown). it seems that His-Tag displayed on the flagella is enough to do its work.
  • On the other hand, the deletion of MotB turned out to be vital for sticking the tagged flagella together.
  • In the presence of FliC-His, cobalt ion adsorb bacteria stronger than nickel ion, this was more or less the expected result.



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)

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