Chiba/Engeneering Flagella

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Latest revision as of 05:26, 27 October 2007

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

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

⊿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

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)
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 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)

@@ Line 2: / Line 2: @@
 [[Image:chiba_logo.png|center]]
 __NOTOC__
-{| style="border:0;width:100%;" cellpadding="20px" cellspacing="0"
+{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
 | align="center" |
-[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] | [[Chiba/Engeneering_Flagella|Bacteria Linker]] | [[Chiba/Communication|Communication]] | [[Chiba/Quorum_Sensing|Size Controller]] | [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]] | [[Chiba/Members_Only|メンバ連絡簿]]
+[[Chiba|Home]]<br>
+<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]]
 |}
-==Our Aim==
+==Affinity Tags==
-[[Image:Chiba_stickbacteria.png|frame|'''Fig1.'''Bacteria Linker]]
+===Our Aim===
-To make a stickey hands on ''E.coli'', we focused on their flagella that are located outside of cells. We used 2 following mechanisms:
+[[Image:Chiba_stickbacteria.png|frame|'''Fig. 9''' Affinity tags.]]
-*Display peptides in flagellar fillament.
+To make affinity tags on ''E.coli'', we focused on their flagella that are located outside the cells. We used the following mechanisms:
-*The imidasol group in histidines make a complex bond with metal ions.
+*Display sticky peptides in flagellar filament.
-We combined these two and made a His-tagged flagella (hoping for ? in the hope to ?) stick together via metal ions.
+*His-tag. The imidazole group in histidines make a complex with metal ions.
- 大腸菌同士を吸着させるため，我々は（細胞の外側に突出しているもののうち）鞭毛に着目した．
+We combined these two and made a His-tagged flagella in the hope to stick them together via metal ions.
-==About flagella==
+===[http://www.npn.jst.go.jp/index.html About flagella]===
-''E.Coli'' have 5-10 flagella randomly distributed around the cell.  Most of the time their flagella rotate in one direction(counterclockwise) and the cell swims in a rather straight line.Intermittently, the flagella rotate in opposite direction or pause, as a result the cell turns or tumbles(depending to the number of flagella that do so)Bacterial 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 10-15 μm long,23 nm diameter.It is built from ~20000 subunits of a ~55kDa single protein, flagellin.In the case of E.Coli, there is only one flagellin, FliC.
+''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.
-FliC has three or four domains,D0,D1,D2,D3,and 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)
+''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].
- 大腸菌は5~10本/細胞の鞭毛を持つ．鞭毛を回転/逆回転させることにより環境の良い場所へとtaxisする．
+===="Variable" FliC D3 domain====
- 鞭毛は長さ約10~15μm，半径約23nmの空孔のチューブ状である．（図：菌全体）
+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]
- 鞭毛のフィラメント部はFliCという蛋白質が規則正しく配列した多量体である．（図：鞭毛アップ）
- FliCはD0,D1,D2,D3ドメインを持つ．(種類によってはD1,D2,D3の３つのドメインを持つ)
- D1とD2はformation of the functional flagellar formation(ffff)の為に必要であるが，D3ドメインは必要でなく可変である（図：蛋白質構造＆鞭毛断面図）
+===About Histidine Tag===
+See [http://en.wikipedia.org/wiki/His-tag wikipedia article].
-==="Variable" FliC D3 domain===
+==Experiments==
-It is possible to display proteins(up to 49.4kDa)on the cell surface of E.Coli using flagellin fusion protein.
+===[[Chiba/Flagella/Making_FliC-his|Making ''FliC-his'' gene]]===
- 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．
+*We inserted the short peptide with six histidine (“His-Tag”) into the fliC D3 domain.
+[[Image:Chiba_flichisgene.png|frame|'''Fig.10''' flic his gene]]<br>
+*[[Image:Chiba_check.png]] Sequence Confirmed<br>
+*[[Image:Chiba_check.png]] Swarm Confirmed<br>
+*[[Image:Chiba_check.png]] Flagella strained with anti-flagella antibody
-References
+===Checking the "Stickiness": Beads Adsorption===
-#Kuwajima, G. ''et al''.: Nature Biotechnology, 6, 1080-1083 (1988).
-#Tanskanen, J. ''et. al''.: Appl. and Env. Microbiol., 4152-4156 (2000)
-==Parts Assembly==
-===[[Chiba/Flagella/Making_FliC-his|FliC-hisの作成]]===
-*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”).
-#Linker Ligation
-*完成プラスミド
-[[Image:FliC His-tag.jpg|frame|left|fig1]]<br clear="all">
-*check Phenotype
-#SDS-PAGE
-#Western-Blotting
-*Display check
-#Beads adsorption
-===[[Chiba/Flagella/FliC-his_generator|FliC-His generator]]===
-*His-tagを入れたFliCをpLuxの下に置き、LuxRが発現されている条件のもとならば、Quorum SensingでFliCを発現させることができるようにする。
-*Quorum Sensingのための遺伝子回路がcolEI oriのvectorに乗っているために、p15Aのベクターを使いdouble transformation することでQuorum Sensingと合わせることができるようになる。
-*pLuxをもつベクター側とFliCをPCRを使って、Ligationさせることで作る。
-===[[Chiba/Flagella/FliC-His_Biobrick|FliC-his biobrick]]===
-必要なこと
-*puc19 vectorの乗っているのでbiobrickのベクターに乗せる。
-*制限酵素サイト(EcoRI,SpeI,PstI)をつぶす。
-*FliC His-TagにはEcoRI,SpeI,PstIが含まれているために、そのままではvectorに入れられない。
- #片側をblunt end もう一方をApaIの制限酵素サイトをつけ、PCRする。
- #vector側も同様にPCRしLigationさせる。
-==Experiments==
-===[[Chiba/Flagella/Display_Check|Display Check: Beads Adsorption]]===
 ====Purpose====
-FliC-hisが発現され,鞭毛にHistidine-Tagがディスプレイされてるか,を確認する．
+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.
-To confirm that whether Histidine-Tag protein were displaied on the flagella                       surface of E.Coli
+====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
+<br>
-====Method====
+====Testing Procedure====
-[[Image:Chiba_fla_beadsintro.png|frame|fig1]]
+#pUC19-FliC-His was transformed to JW1908(''fliC''), JW0747(''MotB''), and GI826(''fliC'' ''motB'').
-ビーズ吸着の説明．
+#Grown to stationary phase
-ヒスチジンはCo（またはNi）と錯体結合?をする．表面に金属イオン（本実験ではCo2+およびNi2+）を配置したビーズを使用して，FliC-hisがディスプレイされた大腸菌とそうでないものを調べた．
+#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.
-ビーズには4つのカルボキシル基のついた炭素鎖がついていて、二価の金属イオンの6つの配位部位のうち、4つと配位結合している。さらに残りの二つの配位部位に、Histidineのイミダゾール基が配位する。このため、Histidine Tagが鞭毛にDisplayされている大腸菌と、ビーズ溶液を懸濁させれば、鞭毛上のHistidineと金属イオンとを媒体として、鞭毛がBeadsに吸着する。この懸濁溶液にMagnetを近づけることにより、BeadsはMagnetに引き寄せられ、Beadsを含まない溶液を上澄みとして分離できる。さらにこのBeadsにImidazoleを含むバッファーを加えれば、このImidazoleがHistidineのImidazole基と競合し、金属イオンにHistidineよりも強い力で配位するため、鞭毛がBeadsからはずれる。ここで再びMagnetを近づければ、大腸菌のついていないBeadsと、大腸菌がいる上澄み溶液とに分離される。この上澄み溶液をプレートにinculateし、コロニーをチェックする。コロニーが形成されれば、鞭毛にHistidine TagがDisplayされているということがわかる。
+====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">
+*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>
-（＜不十分＆不正確です．直してください）
+'''2.''Stickiness'' check using MotB strain'''
+[[Image:Chiba Beads-Adsorption result2.png|frame|left|'''fig. 12''' Binding test using Strain JW0747(''⊿motB'').]]<br clear="all">
+*Mot B deletion provides cell with the flagella completely assembled but not rotating. <br>
+*'''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.
+*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.
-====Samples====
-*GI826(⊿FliC⊿MotB株)
-**pUC19-FliC-His
-**plasmidなし
-*GI826(⊿FliC⊿MotB株)
-**pUC19-FliC-his
-**plasmidなし
-====Procedure====
-*pUC19-FliC-His,をJW1908(Keio⊿FliC株)にトランスフォーメーション。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、inculateする。(Negative Control 1:Noplasmid,Negative Control 2:Co2+あるいはNi2+の有り・無し) →吸着していなかった
-*pUC19-FliC-His,をGI826(⊿FliC⊿MotB株)にトランスフォーメーション。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、inculateする。(Negative Control 1:Noplasmid,Negative Control 2:Co2+あるいはNi2+の有り・無し) →吸着していた。
-====Results====
+<!--
-[[Image:Beads-Adsorption Colony-Number.jpg|frame|left|fig2]]<br clear="all">
+*'''FliC(hisなし)はないんでしたっけ？古'''
-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。
+**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
-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。
+*'''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'''
-The number of colony dramatically decreased with out Co2+ or FliC-His plasmid.
+**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。
+-->
-[[Image:Chiba_fla_beads.png|frame|left|fig3]]<br clear="all">
+==References==
+. Kuwajima, G. ''et al''.: ''J. Bacteriol.'', '''170''', 3305-3309 (1988)<br>
+. Ezaki, S. ''et. al''.: ''J. Ferment. Bioeng.'', '''86''', 500-503 (1998)<br>
+. Baba, T. ''et. al''.: ''Mol. Systems. Biol.,'' '''21''', 1-10 (2006)<br>

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