Difference between revisions of "Part:BBa K1463604"
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[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1463600 FliC] with [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0032 B0032] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 B0034] RBS under the control of [https://parts.igem.org/wiki/index.php?title=Part:BBa_J23100 J23100] promoter.
 
[https://parts.igem.org/wiki/index.php?title=Part:BBa_K1463600 FliC] with [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0032 B0032] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034 B0034] RBS under the control of [https://parts.igem.org/wiki/index.php?title=Part:BBa_J23100 J23100] promoter.
  
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<br> This composite part was made by inserting a synthesised double-stranded oligonucleotide containing [https://parts.igem.org/wiki/index.php?title=Part:BBa_J23100 J23100] promoter (strong) and [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0032 B0032] into [https://parts.igem.org/Part:BBa_K1463601 K1463601] (fliC and B0034 RBS).
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This composite part was made by inserting a synthesised double-stranded oligonucleotide containing [https://parts.igem.org/wiki/index.php?title=Part:BBa_J23100 J23100] promoter (strong) and [https://parts.igem.org/wiki/index.php?title=Part:BBa_B0032 B0032] into [https://parts.igem.org/Part:BBa_K1463601 K1463601] (fliC and B0034 RBS).
  
 
As shown in Figure 1D, this construct in pSB1C3 failed to restore swimming in knockout fliC strains. On sequencing we found this plasmid to have a mutation in the promoter, explaining this result. We failed to clone a functional J23100 promoter in front of the fliC biobrick, suggesting that strong over expression of fliC may be toxic.
 
As shown in Figure 1D, this construct in pSB1C3 failed to restore swimming in knockout fliC strains. On sequencing we found this plasmid to have a mutation in the promoter, explaining this result. We failed to clone a functional J23100 promoter in front of the fliC biobrick, suggesting that strong over expression of fliC may be toxic.
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<br><b>(C)</b> DS941 ΔfliC + pSB1C3 fliC (no promoter), <b>(D)</b> DS941 ΔfliC + J23100 (mutant promoter) fliC, <br><b>(E)</b> DS941 ΔfliC + J23116-fliC(1), <b>(F)</b> DS941 ΔfliC + J23116-fliC(2), <br><b>(G)</b> DS941 ΔfliC + J23106-fliC(1), <b>(H)</b> DS941 ΔfliC + J23106-fliC(2)</p>
 
<br><b>(C)</b> DS941 ΔfliC + pSB1C3 fliC (no promoter), <b>(D)</b> DS941 ΔfliC + J23100 (mutant promoter) fliC, <br><b>(E)</b> DS941 ΔfliC + J23116-fliC(1), <b>(F)</b> DS941 ΔfliC + J23116-fliC(2), <br><b>(G)</b> DS941 ΔfliC + J23106-fliC(1), <b>(H)</b> DS941 ΔfliC + J23106-fliC(2)</p>
  
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<br>
 
 
https://static.igem.org/mediawiki/2014/f/fc/GU_Figure_2_Motility_histogram.png
 
https://static.igem.org/mediawiki/2014/f/fc/GU_Figure_2_Motility_histogram.png
 
<br><b>Figure 2 - FliC Motility Histogram</b> The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive see K1463604.
 
<br><b>Figure 2 - FliC Motility Histogram</b> The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive see K1463604.
  
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<br><br>
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For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#fliC
 
For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#fliC
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
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|}.
 
|}.
  
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===Experiences===
  
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===Proof the FliC protein production===
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====Proof the FliC protein production====
 
We investigate if the FliC protein was well produced by our biobrick.  
 
We investigate if the FliC protein was well produced by our biobrick.  
  
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The FliC size we were looking for was 51,3kDA.
 
The FliC size we were looking for was 51,3kDA.
  
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===Proof of swimming recovery===
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====Proof of swimming recovery====
  
 
[[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 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]]
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<i>.</i>
 
<i>.</i>
  
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===Flagellum features by electronic microscopy===
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====Flagellum features by electronic 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]]
 
[[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
 
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.
 
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.
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==Improvement of the biobrick [https://parts.igem.org/Part:BBa_K1463604 K1463604]==
 
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This biobrick has been improved from a previous one designed by Glasgow 2014 team. Please find the link of this biobrick below :
 
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[https://parts.igem.org/Part:BBa_K1463604 K1463604]
 
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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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===Usage and Biology===
 
===Usage and Biology===
  
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==Sequence and Features==
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1463604 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1463604 SequenceAndFeatures</partinfo>
  

Revision as of 19:49, 15 October 2016

Failed BBa_K1463604

FliC with B0032 and B0034 RBS under the control of J23100 promoter.

This composite part was made by inserting a synthesised double-stranded oligonucleotide containing J23100 promoter (strong) and B0032 into K1463601 (fliC and B0034 RBS).

As shown in Figure 1D, this construct in pSB1C3 failed to restore swimming in knockout fliC strains. On sequencing we found this plasmid to have a mutation in the promoter, explaining this result. We failed to clone a functional J23100 promoter in front of the fliC biobrick, suggesting that strong over expression of fliC may be toxic.

Flic Motility Swarm Assay

310px-GU_Figure_1_swarm_M.png

Figure 1: FliC Swarm Motility Assays.
(A) DS941, (B) DS941 ΔfliC,
(C) DS941 ΔfliC + pSB1C3 fliC (no promoter), (D) DS941 ΔfliC + J23100 (mutant promoter) fliC,
(E) DS941 ΔfliC + J23116-fliC(1), (F) DS941 ΔfliC + J23116-fliC(2),
(G) DS941 ΔfliC + J23106-fliC(1), (H) DS941 ΔfliC + J23106-fliC(2)

GU_Figure_2_Motility_histogram.png
Figure 2 - FliC Motility Histogram The promoters indicated in the histogram were used to drive the fliC biobrick K1463600 with the RBS B0034. Plasmids containing these constructs were used to complement a chromosomal fliC mutation. The diameter of swimming in a 16 hour swarm assay at 37 degrees is shown. The error bars indicate the range or results obtained in two repeats of the experiment. The strong J23100 promoter in this result contained a mutation rendering it inactive see K1463604.


For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#fliC