Chiba/Engeneering Flagella
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__NOTOC__ | __NOTOC__ | ||
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- | [[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]]<br> | + | [[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]] | [[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]] | ||
|} | |} | ||
- | == | + | ==Affinity Tags== |
===Our Aim=== | ===Our Aim=== | ||
- | [[Image:Chiba_stickbacteria.png|frame|'''Fig. 9''' | + | [[Image:Chiba_stickbacteria.png|frame|'''Fig. 9''' Affinity tags.]] |
- | To make | + | 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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==Experiments== | ==Experiments== | ||
- | ===[[Chiba/Flagella/Making_FliC-his|Making | + | ===[[Chiba/Flagella/Making_FliC-his|Making ''FliC-his'' gene]]=== |
*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. | ||
- | [[Image:Chiba_flichisgene.png]]<br> | + | [[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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====Testing Procedure==== | ====Testing Procedure==== | ||
- | #pUC19-FliC-His was transformed to JW1908(''fliC''), | + | #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==== | ||
'''1.''Stickiness'' check using FliC strain''' | '''1.''Stickiness'' check using FliC strain''' | ||
- | [[Image:Chiba Beads-Adsorption result3.png|frame|left| | + | [[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! | *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> | *We thought the problem might be the super-fast revolution of flagella itself. We decided to try MotB strain.<br><br><br> | ||
'''2.''Stickiness'' check using MotB strain''' | '''2.''Stickiness'' check using MotB strain''' | ||
- | [[Image:Chiba Beads-Adsorption result2.png|frame|left|'''fig. | + | [[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> | *Mot B deletion provides cell with the flagella completely assembled but not rotating. <br> | ||
- | *This time | + | *'''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. | *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. | *On the other hand, the deletion of MotB turned out to be vital for sticking the tagged flagella together. |
Latest revision as of 05:26, 27 October 2007
Home |
Affinity Tags
Our Aim
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.
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
- 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
- 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)