Difference between revisions of "Part:BBa K1951008:Experience"
(→‎Flagellum observed by electronic microscopy)
 
(19 intermediate revisions by 2 users not shown)
Line 1: Line 1:
  
−
__NOTOC__
 
−
 
−
This experience page is provided so that any user may enter their experience using this part.<BR>
 
−
Please enter how you used this part and how it worked out.
 
  
 
==Demonstration of FliC protein production==
 
==Demonstration of FliC protein production==
  
 +
[[File:T--Aix-Marseille--comassiefliC.png|300px|right|center|thumb|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.  
 
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 <b>Claire fill this in</b>.
+
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.  
 
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.  
 
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.  
 
After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer.  
−
<b>Claire link to your protocol page and what heating did you do</b>.
+
 
 +
[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.  
 
The mixture was loaded onto a polyacrylamide gel and migrated during 50min at 180V.  
 
Staining was done using coomassie blue.
 
Staining was done using coomassie blue.
Line 19: Line 20:
 
The FliC is at mass 51,3kDa, and can be clearly seen in the gel photograph.
 
The FliC is at mass 51,3kDa, and can be clearly seen in the gel photograph.
  
−
==Proof of swimming recovery==
+
==Demonstration of motility complementation==
  
−
[[File:T--Aix-Marseille--swim.jpeg|400px|right|center|thumb| We investigated here if swimming was recovered by a knockout FliC strain complementation with our biobrick on soft LB agar gelose. <i> Escherichia coli</i> 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]]
+
[[File:T--Aix-Marseille--swim.jpeg|400px|right|center|thumb|  
 +
We investigated if swimming motility was recovered by a knockout FliC strain.
 +
To test complementation with our biobrick strains were stabbed into soft (<b>Claire check</b> 0.5%) LB agar plates,
 +
and incubated at 37°C for 4 hours.
 +
Three strains are shown:
 +
<i> Escherichia coli</i> 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).  
 +
]]
  
−
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.
 
  
 +
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.
  
−
* 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.
 
  
  
Line 35: Line 51:
  
  
−
<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.
 
  
  
Line 45: Line 58:
 
<i>.</i>
 
<i>.</i>
  
−
==Flagellum features by electronic microscopy==
+
==Flagellum observed by electron microscopy==
−
[[File:T--Aix-Marseille--microscopy.jpeg|400px|left|thumb|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 <u>presence of big and numerous flagellums</u> in the complemented mutant while no any flagellum has been observed in the fliC mutant and less in the WT strain.
+
[[File:T--Aix-Marseille--microscopy.jpeg|400px|left|thumb|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 <i>E.coli</i> 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.
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<i>.</i>
  
 
==Improvement of the biobrick [https://parts.igem.org/Part:BBa_K1463604 K1463604]==
 
==Improvement of the biobrick [https://parts.igem.org/Part:BBa_K1463604 K1463604]==
−
This biobrick has been improved from a previous one designed by Glasgow 2014 team. Please find the link of this biobrick below :
+
 
 +
This biobrick is an improvement on the biobrick designed by the Glasgow 2014 team.  
 
[https://parts.igem.org/Part:BBa_K1463604 K1463604]
 
[https://parts.igem.org/Part:BBa_K1463604 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.
+
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 <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.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 13:01, 19 October 2016


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.






.

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.











.

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.

User Reviews

UNIQadb0dbcb5ccd763d-partinfo-00000000-QINU UNIQadb0dbcb5ccd763d-partinfo-00000001-QINU