Part:BBa_K1463604

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

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)

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

Improvement by BBa_K1951008 which perfectly work

This biobrick has been improved from a previous one designed by Glasgow 2014 team.

Mesure of the bacterial motility (y axe left) in function of the incubation time (x axe) and density of swimmer cells (y axe right) in different backgrounds. Density is based on a scale from 0 to 3 : 0=transparent, 1=slighty turbidity, 2=medium turbidity, 3=high turbidity

• For this biobrick design, Glasgow team results has shown that their FliC wasn't able to recover the swimming capacity showing a bad quality of the synthetised flagellin protein. Please find the link of this biobrick below :

BBa_K1463604Promotor they have used had a mutation which makes it unfunctionnal.

Improved design

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.

Sequence optimization

We took fliC sequence from Glasgow 2014 team BBa_K1463601. We used Snapgene to remove the forbidden site by changing in the substitution which didn't change the amino acid. The sequence has been sythetised by IDT. We changed a Bbs1 site 130 position by substitution of amino acid coding.

Optimized codons for Escherichia coli.

We obtimised codon for Escherichia coli. Codon optimization is a technique used to improve the protein expression in living organism by increasing the translational efficiency of gene of interest [1-4, 6-13, 15-18, 20-28]. This biobrick codon optimised increase the functionality of gene. To process it, we use codon optimization IDT software. If you use this biobrick in Escherichia coli, you can be sure that the protein produced will be highly expressed and well solubilised.

Prefix and suffix addition

Prefix and suffix subsequences (containing restriction site EcoRI, XbaI and SpeI PstI respectively) have been added by a SLIC method with the following oligos :

FliC E. coli slic forward	cgctaaggatgatttctgGAATTCGCGGCCGCTTCTAGATGGCACAAGTCATTAAT
FliC E.coli slic reverse	ttgcccttttttgccggaCTGCAGCGGCCGCTACTAGTATTATTAACCCTGGAGCAG

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Experiences

Proof the FliC protein production

We investigate if the FliC protein was well produced by our biobrick.

To make it, we did a SDS page and comassie blue. From an over night starter, cells have been growth from Abs(600nm)=0.2 until Abs(600nm)=0.6. Then 1uDO has been sampled and centrifuged at 5000g during 5min. After removal of the supernatant, cells were resuspended in 50µL SDS-PAGE sample buffer (Morris formulation). Mix has been charged on a polyacrylamide gel and migrated during 50min at 180V. Revelation was done using comassie blue.

The FliC size we were looking for was 51,3kDA.

Proof of swimming recovery

We investigated here if swimming was recovered by a knockout FliC strain complementation with our biobrick on soft LB agar gelose. Escherichia coli 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

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.

• To test it, we made a fliC mutant of E.coli W3110 strain by transduction using phage P1 [http://2016.igem.org/Team:Aix-Marseille/Experiments/Protocols#.23Protocol_5_:_Generalised_transduction_using_phage_P1 (Transduction protocol)]).

• 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.

Result : 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.

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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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1306
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 383
  Illegal AgeI site found at 791
  
• 1000
  COMPATIBLE WITH RFC[1000]