Difference between revisions of "Team:Aix-Marseille/Results"
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Sandra Youpi (Talk | contribs) |
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- <i>E. coli</i> TG1 strain with pSB1C3 containing the RFP coding sequence (negative control) | - <i>E. coli</i> TG1 strain with pSB1C3 containing the RFP coding sequence (negative control) | ||
| − | - <i>E. coli</i> TG1 strain with | + | - <i>E. coli</i> TG1 strain with des operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011) before and after induction. (You can observe the production of the 4 proteins on the figure on the right; left : before induction, right : after induction) |
Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway. | Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway. | ||
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=== Proof of fonctionnality=== | === Proof of fonctionnality=== | ||
We investigated if DesA (Lysine decarboxylase) was fonctionnal by measurement of cadaverine using HPLC with C18 column and we proved that our biobrick allows the production of DesA which enables to produce the cadaverine and makes it fonctionnal. | We investigated if DesA (Lysine decarboxylase) was fonctionnal by measurement of cadaverine using HPLC with C18 column and we proved that our biobrick allows the production of DesA which enables to produce the cadaverine and makes it fonctionnal. | ||
| − | [[File:T--Aix-Marseille--result5.jpeg|500px|tight|thumb| Investigation of the cadaverine production by the lysine decarboxylase DesA. The cadaverin production has been detected by HPLC using C18 column after induction of the strain. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), cadA mutant from Keio bank (blue column), complemented cadA mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004 Bba_K1951004](orange column), complemented cadA mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004Bba_K1951011(grey column).]] | + | [[File:T--Aix-Marseille--result5.jpeg|500px|tight|thumb| Investigation of the cadaverine production by the lysine decarboxylase DesA. The cadaverin production has been detected by HPLC using C18 column after induction of the strain. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), cadA mutant from Keio bank (blue column), complemented <i> cadA </i> mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004 Bba_K1951004](orange column), complemented <i>cadA</i> mutant from Keio bank with [http://parts.igem.org/Part:BBa_K1951004Bba_K1951011(grey column).]] |
Results showed cadaverine detection in the wild type meaning the original strain well produces the cadaverine. | Results showed cadaverine detection in the wild type meaning the original strain well produces the cadaverine. | ||
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===Proof of swimming recovery=== | ===Proof of swimming recovery=== | ||
| − | We have made a biobrick BbaK1951008 ables to produce Flagellin (FliC protein of the flagellum). In the aim to test the flagellin integrity, we made a fliC mutant in a <i>E.coli | + | We have made a biobrick BbaK1951008 ables to produce Flagellin (FliC protein of the flagellum). In the aim to test the flagellin integrity, we made a <i>fliC</i> mutant in a <i>E. coli</i> W3110 strain by transduction using phage P1 (protocol available on our website). |
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| − | [[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout | + | [[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout <i>fliC</i> strain. |
To test complementation with our biobrick strains were stabbed into soft (0.3%) LB agar plates, | To test complementation with our biobrick strains were stabbed into soft (0.3%) LB agar plates, | ||
and incubated at 37°C for 4 hours. | and incubated at 37°C for 4 hours. | ||
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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. | 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. | + | The image shows mutiple polar flagella in an <i>E. coli fliC</i> 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. |
===In the futur=== | ===In the futur=== | ||
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The improvement of this part is multiple. | 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. | * 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 <i>E.coli</i>. Thus as an expression part is improved over the initial design. | + | * Second the sequence that we have used it a synthetic gene with codon optimisation, designed specifically for high level expression in <i>E. coli</i>. 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. | * 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. | ||
