Part:BBa_K1951008:Experience

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Proof the FliC protein production

We investigate if the FliC protein was well produced by our biobrick.

To make it, we did a SDS page and comassie blue. From an over night starter, cells have been growth from Abs(600nm)=0.2 until Abs(600nm)=0.6. Then 1uDO has been sampled and centrifuged at 5000g during 5min. After removal of the supernatant, cells were resuspended in 50µL SDS-PAGE sample buffer (Morris formulation). Mix has been charged on a polyacrylamide gel and migrated during 50min at 180V. Revelation was done using comassie blue.

The FliC size we were looking for was 51,3kDA.

Proof of swimming recovery

We investigated here if swimming was recovered by a knockout FliC strain complementation with our biobrick on soft LB agar gelose. Escherichia coli W3110 strain has been used as a wild type because of its good swimming capacity. We tested 3 background: W3110 (down left) knockout W3110 fliC mutant(down right) and knockout W3110 fliC mutant complemented with BBa_K1951008(up). Cells were ensemenced using a tooth pic and incubate at 37°C. Photo was taken after 4h incubation

Here, we had a look if the protein produced by our biobrick was well traducted et abled to recover swing in a FliC deficient strain.

• To test it, we made a fliC mutant of E.coli W3110 strain by transduction using phage P1 [http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_5_:_Generalised_transduction_using_phage_P1 (Transduction protocol)]).

• fliC mutant W3110 has been complemented by our biobrick Bba_K1951008. On the following figure, we investigated if the complemented fliC mutant recovered swimming after complementation. To do this, we did a swimming test using soft gelose.

Result : WT W3110 strain was well swimming after 3 hours incubation at 37°C. However, the FliC mutant motility was absent whereas fliC mutant complemented recover the swimming ability, making a proof that our biobrick is produced a fonctionnal flagellin protein. Moreover, the circle was more intense in the complemented strain than in the wild type showing that more bacteria were able to swim.

Complementation by transformation of the W3110 mutant recovered the swimming capacity showing the integrity of the flagellin produced by Bba_K1951008.

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Flagellum features by electronic microscopy

Electronic microscopy from fliC mutant complemented by BBa_1951008. From an over night starter, culture was started and a sample of 1uDO was taken after 3/4hours incubation at 37°C under agitation and observed by electronic microspcopy using negative analysis. Scale: 600nm/cm

This step aims to observe the good assembly of the flagellin protein

We analysed the fliC mutant made by transduction as describe before complemented by BbaK1951008 using electronic microscopy with negative filter. This tools allowed us to observe the flagellum integrity recovered and to obtain image of our work. Image shows the presence of big and numerous flagellums in the complemented mutant while no any flagellum has been observed in the fliC mutant and less in the WT strain.

Improvement of the biobrick K1463604

This biobrick has been improved from a previous one designed by Glasgow 2014 team. Please find the link of this biobrick below : K1463604

Instead of Bba_J23106 and Bba_J23116, we used strong promoter, strong RBS combination for high expression levels of the flagellin. By the combination of Bba_K880005 and Bba_K1951005, we made a high flagellin expression vector able to highly recover swimming and even surexpress this pattern.

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