Difference between revisions of "Part:BBa K1951008:Experience"

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Demonstration of FliC protein production

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 Claire fill this in. 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. Claire link to your protocol page and what heating did you do. 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.

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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 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.
-* To test it, we made a fliC mutant of <i>E.coli W3110</i> strain by transduction using phage P1 [http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_5_:_Generalised_transduction_using_phage_P1 (Transduction protocol)]).
+We made the fliC deletion mutant of <i>E.coli</i> 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.
-* 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.
-<u>'''Result'''</u> : 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.
-<i>.</i>
 ==Flagellum features by electronic microscopy==