Part:BBa_K1951008:Experience

Demonstration of FliC protein production

Test of our biobrick proteins production using a SDS page and comassie

We investigated if the FliC protein was well produced by our biobrick using SDS PAGE.

To to do this we performed SDS PAGE and stained with coomassie blue using cells containing this biobrick in plasmid backbone [http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.2312_:_SDS_page_and_coom SDS page and coomassie blue]

From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=0.6.

Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min.

After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.

[http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.2312_:_SDS_page_and_coom SDS page and coomassie blue] The mixture was loaded onto a polyacrylamide gel and migrated during 50min at 180V. Staining was done using coomassie blue.

The FliC is at mass 51,3kDa, and can be clearly seen in the gel photograph.

Demonstration of motility complementation

We investigated if swimming motility was recovered by a knockout FliC strain. To test complementation with our biobrick strains were stabbed into soft (Claire check 0.5%) LB agar plates, and incubated at 37°C for 4 hours. Three strains are shown: Escherichia coli W3110 wild-type strain, which has a good swimming capacity (lower right); a fliC deletion mutant of W3110 (lower left); and the fliC mutant complemented with BBa_K1951008(top).

In the figure the deletion mutant (lower left sector) shows no swimming motility as expected and a small white colony. In contrast the wild-type colony (lower right) has a diffuse halo due to swimming cells around the central white colony. Finally the complemented strain, the deletion mutant complemented with our biobrick, (top panel) shows two colonies with intense halos surrounding them. This illustrated clearly that our biobrick can restore motility and is functional. The intensity of the halo suggests that a greater proportion of the cells are mobile or swimming is in someway better than the wild-type.

We made the fliC deletion mutant of E.coli W3110 by transduction using the phage P1 [http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_5_:_Generalised_transduction_using_phage_P1 (Transduction protocol)]), using as a source the mutation from the Keio collection (http://cgsc.biology.yale.edu/KeioList.php)

We conclude that this biobrick if fully functional producing a flagellin protein that can be incorporated into functional flagella and so rescue the non-motile deletion mutant.

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Flagellum observed by electron microscopy

Electron micrograph of the fliC deletion mutant complemented by BBa_1951008. Cells were deposited onto carbon coated electon microscope grids and negativly stained using uranyl acetate. Scale bar : 600 nm

This check the flagella assembly and the integration of the flagellin protein expressed from our biobrick BBa_K1951008 we have observed bacteria with an electron microscope.

The image shows mutiple polar flagella in an E.coli fliC deletion mutant containing our biobrick. We saw that these cells had more flagella than wild-type (W3110) cells and that the fliC deletion mutant did not have flagella.

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Improvement of the biobrick K1463604

This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team. K1463604

The improvement of this part is multiple.

• First there is no mutation in the promotor or RBS of our part so the flagellin is well expressed and functional. Unfortunately when the Glasgow team trie to make this part they picked up a mutation in the promotor.
• Second the sequence that we have used it a synthetic gene with codon optimisation, designed specifically for high level expression in E.coli. Thus as an expression part is improved over the initial design.
• Third in the functional swimming assay, we see evidence for improved swimming (denser halo), in cells expressing our biobrick, a phenotype not observed by the Glasgow team in 2014.

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