Chiba/Engeneering Flagella
From 2007.igem.org
(→Results&Discussion) |
(→Results&Discussion) |
||
Line 84: | Line 84: | ||
2.Strainの比較 | 2.Strainの比較 | ||
[[Image:Chiba Beads-Adsorption result2.png|frame|left|fig2. Strain JW1908(''⊿motB'').]]<br clear="all"> | [[Image:Chiba Beads-Adsorption result2.png|frame|left|fig2. Strain JW1908(''⊿motB'').]]<br clear="all"> | ||
+ | <br> | ||
+ | *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 |
Revision as of 01:37, 27 October 2007
Introduction | Project Design ( 1.Sticky Hands | 2.Communication | 3.Size Control ) | Making Marimos | Our Goal || Team Members | メンバ連絡簿 |
Stickey Tags
Our Aim
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.
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[1].
"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.[2]
References
- Kuwajima, G. et al.: Nature Biotechnology, 6, 1080-1083 (1988).
- Tanskanen, J. et. al.: Appl. and Env. Microbiol., 4152-4156 (2000)
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-tag are displaied on the flagella and are capable of binding to Co2+-surface.
Samples
- JW1908(⊿fliC strain)
- pUC19-fliC-his
- no plasmid
- GI826(⊿fliC⊿motB strain)
- pUC19-fliC-his
- no plasmid
Procedure
- pUC19-FliC-His was transformed to JW1908(fliC) and GI826(fliC motB).
- Incubated 4 cell samples written above.
- Culture was suspended with beads which will stick to histidine.
- Adsorbed E.coli on the beads was washed with a buffer and spreaded on agar plates.
- The colony of each plate was counted.
Results&Discussion
1.Stickiness check
- In the presence of Co2+Histidine tag,beads
Co2+Histidine Tagの存在よって大腸菌がビーズに吸着している.
- 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
- 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