Difference between revisions of "Team:Aix-Marseille/Results"

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{{:Team:Aix-Marseille/Template-Top|Result}}
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{{:Team:Aix-Marseille/Template-Top| Results}}
  
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Our lab experience enabled us to create the final Biobrocks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
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Our lab experience enabled us to create the final Biobricks of our project which are a siderophore Desferrioxamine B producer and a flagellin producer.
  
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==MObilisation result==
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==Mobilisation results==
  
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===Proof of protein production===
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===Protein production===
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Production of every enzymes involved in the pathway has been shown by a SDS page and Comassie Blue. We compared the production of proteins in different background :
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- <i>E. coli</i>  Tg1 strain without any plasmide
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[[File:T--Aix-Marseille--result7.jpeg|500px|right|thumb|Test of our biobrick proteins production using a SDS PAGE and coomassie blue staining. - : no induction ; + : induction with 0.02% arabinose at Abs(600nm)=0.4]]
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We investigated if the DesA, DesB, DesC and DesD proteins were well produced by our biobrick [http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011] using SDS PAGE.
  
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- <i>E. coli</i>  Tg1 strain with pSB1C3 containing the RFP coding sequence
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To do this we performed SDS PAGE [https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Protocol_.2312_:_SDS_page_and_coomassie_blue (protocol)] and stained with coomassie blue using cells containing this biobrick. From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=1.
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Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min.
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After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.
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We heated the mix at 95°C during 15min.
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The sample was loaded onto a polyacrylamide gel and migrated during 50min at 180V.
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Staining was done using coomassie blue.
  
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- <i>E. coli</i>  Tg1 strain complemented with our biobrick Bba_K1951011 before and after induction. (you can observe the production of the 4 proteins on the figure below on the left; right : before induction, left : after induction)
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We compared the production of proteins in different background :
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- <i>E. coli</i>  TG1 strain with pSB1C3 containing the RFP coding sequence (negative control)
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- <i>E. coli</i>  TG1 strain with <i>des</i> 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)
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Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway.
  
 
=== Proof of fonctionnality===
 
=== Proof of fonctionnality===
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We investigated if DesA (Lysine decarboxylase) was fonctionnal, by measurement of cadaverine using HPLC with C18 column and proofed our biobrick is a producer of this protein and makes it fonctionnal.   
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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.   
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[[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 <i>des</i> genes. Different backgrounds were analysed : wild type <i>Escherichia coli</i> TG1 strain (yellow column), <i>cadA</i> mutant from Keio bank (blue column), complemented <i> cadA </i> mutant from Keio bank with p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 BBa_K1951004]) (orange column), complemented <i>cadA</i> mutant from Keio bank with <i>des</i> operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011]) (grey column).]]
  
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[[File:T--Aix-Marseille--result1.jpeg|1000px|center|thumb|<u>On the right</u>, we tested the protein production using a SDS page and comassie in a <i>Escherichia coli</i> Tg1 strain after transformation with Bba_K1951011. On the right column, it has been observed the protein production without induction with 0.02% arabinose. On the left column, protein production was tested after induction. Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine biosynthesis pathway. <u> On the left, </u> we had a look of the cadaverine production by the lysine decarboxylase DesA. Production has been detected by HPLC using C18 column after induction of the strain. <i>Escherichia coli</i> Tg1 strain was used as a wild type. Cadaverine production has been detected in this strain. In a Tg1 cadA mutant, cadaverine was also produced in a least quantity showing that an other pathway is responsible for the production of cadaverine. In the cadA mutant complemented by Bba_K1951004, the amount of cadaverine was recovered and even beyond the wild type production. Moreover, in the cadA mutant complemented by Bba_K1951011, the cadaverine level produced was even over the wild type and complemented Bba_K1951004 production ]]
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Results showed cadaverine detection in the wild type meaning the original strain well produces the cadaverine.
  
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===In the futur===
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In <i>cadA</i> mutant from Keio bank, cadaverine was also produced in a least quantity showing that an other pathway is responsible for the production of cadaverine (existence of an other constitutive lysine decarboxylase in the <i> E. coli </i> genome.
  
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We plan to test the whole pathway viability in the first hand.  
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In the <i>cadA</i> mutant complemented by p<i>desA</i> ([http://parts.igem.org/Part:BBa_K1951004 BBa_K1951004]), the amount of cadaverine was recovered and even beyond the wild type production.
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In a second hand, the potential adsorption of platium by our siderophore would be investigate in a rich metal medium.
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Moreover, in the <i>cadA</i> mutant complemented by the des operon ([http://parts.igem.org/Part:BBa_K1951011 BBa_K1951011]), the cadaverine level produced was even over the wild type and complemented Bba_K1951004 production. We explain this result because of the higher stability of this big composite part.
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To conclude, we made a big composite part able to produce every proteins involved in the biosynthetic pathway of the desferrioxamine B.
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===In the future===
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* Test the whole pathway
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- siderophore production (HPLC and absorption spectroscopy)
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- Improve the siderophore production if needed (cofactors, protein stability, metabolic engineering)
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* Binding affinity for platinum by our siderophore
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- ICPAES (ion coupled plasma atomic emission spectroscopy)
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* Importation of the siderophore in ''Streptomyces coelicolor''
  
 
==Biosorption result==
 
==Biosorption result==
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=== Protein production===
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[[File:T--Aix-Marseille--comassieflic.jpeg|left|400px|thumb|Test of proteins production with biobrick [http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008] using a SDS PAGE and coomassie blue stain]]
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We investigated if the FliC protein was well produced by our biobrick ([http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008]).
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To do this we performed SDS PAGE and stained with coomassie blue using cells containing this biobrick.
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From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=0.6.
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Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min.
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After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.
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The sample was loaded onto a polyacrylamide gel and migrated during 50min at 180V.
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Staining was done using coomassie blue.
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The FliC is at mass 51,3kDa, and can be seen in the gel photograph.
  
 
===Proof of swimming recovery===
 
===Proof of swimming recovery===
  
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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 W3110</i> strain by transduction using phage P1 (protocol available on our website). fliC mutant 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. WT W3110 strain was well swimming after 3 hours incubation at 37°C. However, FliC mutant wasn't able to swim whereas fliC mutant complemented recover the swimming ability, making a proof that our biobrick is production a fonctionnal flagellin protein.  
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We have made a biobrick [http://parts.igem.org/Part:BBa_K1951008 BBa_k1951008] 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 ([https://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#Step2_:_Transduction_P1 protocol]).  
  
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[[File:T--Aix-Marseille--result2.jpeg|1000px|center|thumb|<u>On the right</u>, we investigate if swimming was recovered by a knockout FliC strain complemented by our biobrick. W3110 strain was been used has a wild type because of its good swimming capacity (down left). From this strain, we made a mutant by transduction using phage p1 and W3110 fliC mutant was not able to swin anymore. Complementation by transformation of the W3110 mutant recovered the swimming capacity showing the integrity of the flagellin produced by Bba_K1951008. <u>On the left</u>, we did electronic microscopy. From an over night starter, culture was started and a sample of 1uDO was taken after 3/4hours culture and observed by electronic microspcopy. Image shows the present of flagellums in the complemented mutant while no any flagellum has been observed in the fliC mutant strain]]
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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.  
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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.
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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.
  
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===Microscopie of the flagellum===
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[[File:T--Aix-Marseille--result3.jpeg|770px|center|thumb|We investigated if swimming motility was recovered by a knockout <i>fliC</i> strain.
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To test complementation with our biobrick ([http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008]), strains were stabbed into soft (0.3%) LB agar plates,
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and incubated at 37°C for 4 hours.
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Three strains are shown:
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<i> Escherichia coli</i> W3110 wild-type strain, which has a good swimming capacity (lower right);
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a fliC deletion mutant of W3110 (lower left);
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and the fliC mutant complemented with [http://parts.igem.org/Part:BBa_K1951008 BBa_K1951008] (top).
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]]
  
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We analysed the fliC mutant complemented by BbaK1951008 using electronic microscopy. This tools allowed us to observe the flagellum integrity recovered and to obtain phenotypic image of our work.
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=== Electron microscopy of the flagella===
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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.
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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. The experiments suggest the functionality of our biobrick even if this experiment need a proper negative control i.e. a deletion mutant with no flagella in order to prove effect of our biobrick.
  
 
===In the futur===
 
===In the futur===
  
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We plan to make a proof of concept of the platinum absorption to the surface of the flagellin.
 
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In a second time, we want to finish the insertion of the restriction site to allow the cassette insertion ( peptide absorbing specifically precious metal) and test their affinity.
 
  
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===Improvement of FliC <i>E. coli</i> BBa_K342000===
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* Adsorption of platinum by our flagella
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- election microscopy observation
  
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This biobrick has been improoved from a previous one designed by Glasgow 2014 team. Please find the link of this biobrick below :
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* Finalise the insertion of platinum specific peptide
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http://parts.igem.org/Part:BBa_K1463601
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Instead of promotors 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 recover swimming.
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- directed mutagenesis introducing a Bbs1 restriction site
  
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==Project achievement in a table==
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- insertion of coding sequence of peptide by annealing
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<div class='content_wrapper'>
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<div class='text_box'>
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==Futur plan for our project==
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* Metal recovery by calcination or proteolysis
  
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Now that every biobricks have been created and are available, make proofs of concept could allow to envisage investigation about possibilities of an industrial application. The purely biologic production of high precious metal adsorber could reduce environnemental impact of the precious metal production and its cost while renew essential precious metals like platinum, for a sustainable production.
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* Extend to other precious metals (other peptides : gold, silver, palladium)
  
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[[File:T--Aix-Marseille--igem.jpeg|500px|center|thumb|Igem Aix Marseille 2016 in a sustainable circle]]
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===Improvement of FliC <i>E. coli</i> [http://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]===
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<h5> Project Achievements </h5>
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team.
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[http://parts.igem.org/Part:BBa_K1463604 BBa_K1463604]
  
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<ul>
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The improvement of this part is multiple.
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<li>A list of linked bullet points of the successful results during your project</li>
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* 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.
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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* 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.
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</ul>
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* 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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<h5>Inspiration</h5>
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==Futur plan for our project==
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<p>See how other teams presented their results.</p>
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<ul>
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
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</ul>
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</div>
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Now that every biobrick have been created and are available, make proof of concept could allow to envisage investigation about possibilities of an industrial application.
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</div>
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[[File:T--Aix-Marseille--maquette2.jpeg|770px|center|thumb|Maquette of our process in the futur]]
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The biologic production of high and specific adsorber could reduce environnemental impact of the metal production and its cost while renew essential precious metals like platinum, for a sustainable production.
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[[File:T--Aix-Marseille--igem.jpeg|500px|center|thumb|Igem Aix Marseille 2016 in a sustainable circle]]
  
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Latest revision as of 01:16, 20 October 2016