Chiba/Engeneering Flagella

From 2007.igem.org

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

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)