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.Sticky Hands]] | [[Chiba/Communication|2.Communication]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]] | [[Chiba/Members_Only|メンバ連絡簿]]
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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>
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[[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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|}
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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. 
We combined these two and made a His-tagged flagella in the hope to stick them together via metal ions.
We combined these two and made a His-tagged flagella in the hope to stick them together via metal ions.
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===About flagella===
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===[http://www.npn.jst.go.jp/index.html About flagella]===
''E.Coli'' have 5-10 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.
The flagella is used for swimming and for chemotaxis; the bacteria run when they find attractant, tumble when there is a repellent.
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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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====References====
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#Kuwajima, G. ''et al''.: Nature Biotechnology, 6, 1080-1083 (1988).
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#Tanskanen, J. ''et. al''.: Appl. and Env. Microbiol., 4152-4156 (2000)
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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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*[[Image:Chiba_check.png]] Flagella strained with anti-flagella antibody
*[[Image:Chiba_check.png]] Flagella strained with anti-flagella antibody
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===[[Chiba/Flagella/Display_Check|Checking the "Stickiness": Beads Adsorption]]===
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===Checking the "Stickiness": Beads Adsorption===
====Purpose====
====Purpose====
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Confirm that the his-tag are displaied on the flagella and are capable of binding to Co<sup>2+</sup>-surface.
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Confirm that the his-tags are displaied on the flagella and are capable of binding to Co<sup>2+</sup>- or Ni<sup>2+</sup>- surface.
====Samples====
====Samples====
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*JW1908(⊿fliC strain)
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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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*GI826(⊿fliC⊿motB strain)
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*⊿fliC,⊿motB strain(GI826)transformed with
**pUC19-fliC-his
**pUC19-fliC-his
**no plasmid
**no plasmid
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<br>
 +
====Testing Procedure====
 +
#pUC19-FliC-His was transformed to JW1908(''fliC''), JW0747(''MotB''), 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)
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#E" coli" detached from beads by adding imidazole then spreaded on agar plates.
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#The number of the colonies on resultant plates.
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====Results&Discussion====
 +
'''1.''Stickiness'' check using FliC strain'''
 +
[[Image:Chiba Beads-Adsorption result3.png|frame|left|'''Fig. 11''' Binding test using Strain JW1908(''⊿fliC'',).]]<br clear="all">
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*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.<br><br><br>
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====Procedure====
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'''2.''Stickiness'' check using MotB strain'''
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#pUC19-FliC-His was transformed to JW1908(''fliC'') and GI826(''fliC'' ''motB'').
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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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#Incubated 4 cell samples written above.
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*Mot B deletion provides cell with the flagella completely assembled but not rotating. <br>
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#Culture was suspended with beads which will stick to histidine.
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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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#Adsorbed ''E.coli'' on the beads was washed with a buffer and spreaded on agar plates.
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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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#The colony of each plate was counted.
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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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*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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====Results&Discussion====
 
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1.Stickiness check
 
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[[Image:Beads-Adsorption Colony-Number.jpg|frame|left|fig2. Strain GI826(''fliC'',''motB'').]]<br clear="all">
 
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*In the presence of Co<sup>2+</sup>Histidine tag,beads
 
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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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*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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*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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*The number of colony dramatically decreased with out Co2+ or FliC-His plasmid.
 
 +
 +
<!--
*'''FliC(hisなし)はないんでしたっけ?古'''
*'''FliC(hisなし)はないんでしたっけ?古'''
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**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
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**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'''
*'''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'''
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**
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**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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-->
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2.Strainの比較(データ?)
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{| style="border:1px solid #555555;" cellspacing="0" cellpadding="5"
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| style="border:1px solid #555555;" |
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Strain
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| style="border:1px solid #555555;" |
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Co<sup>2+</sup>
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| style="border:1px solid #555555;" |
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No metal
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|-
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| style="border:1px solid #555555;" |
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JW1908<br>
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''fliC''(-), '''''motB''(+)'''
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| style="border:1px solid #555555;" |
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吸着していない
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| style="border:1px solid #555555;" |
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吸着していない
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|-
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| style="border:1px solid #555555;" |
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GI826<br>
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''fliC''(-), '''''motB''(-)'''
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| style="border:1px solid #555555;" |
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吸着していた
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| style="border:1px solid #555555;" |
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吸着していない
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|}
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*'''ここで言うところの、吸着していた・していない、という差はどんなデータから判断していますか?by とよたろ'''
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==References==
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**stickness checkの結果から判断しました。
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3. Kuwajima, G. ''et al''.: ''J. Bacteriol.'', '''170''', 3305-3309 (1988)<br>
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*冨永くんのデータ記載待ちです.それか今日学校で確認します.古
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4. Ezaki, S. ''et. al''.: ''J. Ferment. Bioeng.'', '''86''', 500-503 (1998)<br>
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**取れているデータはすべて記載してあります。
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5. Baba, T. ''et. al''.: ''Mol. Systems. Biol.,'' '''21''', 1-10 (2006)<br>

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 | [http://chem.tf.chiba-u.jp/igem/ 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.

[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.
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)