Part:BBa_K1617000

Modular Flagellin - FliC_MCS

Flagellin is the filament forming component of bacterial flagella. The up to 20 µm long filaments are formed by the self-assembly of flagellin. Flagellin proteins consist of four domains (D0-D3) and have a size of about 20-30kDa. While the D0 and D1 domains are well conserved, the D2 and D3 domains are highly variable in sequence and length [1]. Here, we created a modular flagellin with an easily replacable D3 region. A multiple-cloning site flanking the D3 domains allows for the design of custom-made flagellins by being compatible with iGEM fusion standards such as RFC25 and RFC21. You can pick any RFC25 compatible BioBrick from the registry and test its functionality immediately via a facile motility assay. This system enables the creation of a three-dimensional reactive nanostructure that offers an increased specific surface with high catalytic activity. With this method produced functionalized flagella filaments can be easily harvested via shearing stress following a simple protocol. Flaggela are self-assembling polymer systems. A temperature increase up to 95 °C depolymerizes the flagella and disconnects the flagellin subunits. By cooling down, these subunits assemble back into fully formed flagella filaments. This means that various flagellin subunits with different molecular set-ups can be combined and used to form multifunctional nanostructures. The resulting flagella may be constructed consisting of various different active sites, which will enable the combination of multiple enzymatic steps in close proximity. FliC MCS is a monomer which is stacked in a helical manner forming the filament of flagella. In this part we used the fliC from Escherichia coli str. K-12 substr. MG1655. As the natural self-assembly involves the transportation of fliC thorugh the filament tube there are steric limitations to the flagellin design. If flagellin-hybrids have a higher molecular mass than the wildtype flagellin (about 52 kDa ProtParam) problems may arise due to steric effects. In this case, we suggest using non-canonical aminoacids (ncAAs) for in-vitro bioconjugation via click reaction. You can read more about it in our wiki or in our D3 Biobrick description.

Figure 1 Video showing the self-assembly of the bacterial flagellum. FliC is the main component which makes up most of the filament. Source: https://youtu.be/GnNCaBXL7LY Figure 2 In the modular flagellin the variable D3 domain was replaced with a polylinker coding a multiple-cloning site. This part may be used to introduce any standard RFC25 part into the flagellin precursor forming novel flagallins.

Usage and Biology

You can use the motility assay to screen for functional flagellin formation.

A: MG1655 z1 ΔFliC B: B = MG1655 z1 C: C = Prom110_FliC_MCS BBa_K1617003

Figure 3 Comparison of the motility between the FliC MCS carrying strain (right) and a negative (left) and positive (middle) control. Since our construct was transformed into E. coli strain MG1655 z1 ΔFliC, we used the highly motile wildtype strain MG1655 z1 as positive control and as negative control the FliC knockout strain MG1655 z1 ΔFliC.

You can create your own functional flagellin. Just use an RFC25 compatible domain, gene region and clone it into the standard FliC MCS using AgeI and NgoMIV. You need to check for the correct alignment though...

Functional Parameters

Design of a functional flaggelin variant which does not contain the variable d3 flagellin domain anymore but instead carries a polylinker region (MCS). This polylinker region is the product of an inserted multi cloning side in place of the endogeneous d3 coding sequence.

Figure 4 Characterization of BBa_K1617003. Through the motility assay it could be shown that our BioBricks work. With cloning the BioBrick into a delta flick deficient strand the motility could be regained. There is no significant difference according the motility between the wildtype and our transformed consturuct in delta flic deficient clones.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 628
  Illegal BamHI site found at 577
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 583
  Illegal AgeI site found at 634
  
• 1000
  COMPATIBLE WITH RFC[1000]

Auto annotator

Protein data table for BioBrick BBa_ automatically created by the BioBrick-AutoAnnotator version 1.0
Nucleotide sequence in RFC 10: (underlined part encodes the protein)  ATGGCACAA ... CTGCAAGGTTAA  ORF from nucleotide position 1 to 1293 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below) 1  101  201  301  401  MAQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDGISVAQTTEGALSEINNNLQRVRELTVQAT TGTNSESDLSSIQDEIKSRLDEIDRVSGQTQFNGVNVLAKNGSAKIQVGANDNQTITIDLKQIDAKTLGLDGFSVKNNDTVTTSAPVTAFGAGSAGVDMG GSMGGSMKLRSTGSGGTPVQIDNTAGSATANLGAVSLVKLQDSKGNDTDTYALKDTNGNLYAADVNETTGAVSVKTITYTDSSGAASSPTAVKLGGDDGK TEVVDIDGKTYDSADLNGGNLQTGLTAGGEALTAVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEAQSRIQDADYATEVSN GSKAQIIQQAGNSVLAKANQVPQQVLSLLQG*
Sequence features: (with their position in the amino acid sequence, see the list of supported features) RFC25 scar (shown in bold):  101 to 102
Amino acid composition: Ala (A) 49 (11.4%) Arg (R) 12 (2.8%) Asn (N) 39 (9.0%) Asp (D) 33 (7.7%) Cys (C) 0 (0.0%) Gln (Q) 27 (6.3%) Glu (E) 12 (2.8%) Gly (G) 42 (9.7%) His (H) 0 (0.0%) Ile (I) 23 (5.3%) Leu (L) 36 (8.4%) Lys (K) 22 (5.1%) Met (M) 4 (0.9%) Phe (F) 5 (1.2%) Pro (P) 5 (1.2%) Ser (S) 45 (10.4%) Thr (T) 43 (10.0%) Trp (W) 0 (0.0%) Tyr (Y) 5 (1.2%) Val (V) 29 (6.7%)
Amino acid counting Total number: 431 Positively charged (Arg+Lys): 34 (7.9%) Negatively charged (Asp+Glu): 45 (10.4%) Aromatic (Phe+His+Try+Tyr): 10 (2.3%)	Biochemical parameters Atomic composition: C1859H3077N555O681S4 Molecular mass [Da]: 44227.4 Theoretical pI: 4.67 Extinction coefficient at 280 nm [M-1 cm-1]: 7450 / 7450 (all Cys red/ox)
Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges
Codon usage Organism: E. coli B. subtilis S. cerevisiae A. thaliana P. patens Mammals Codon quality (CAI): excellent (0.83) good (0.78) good (0.67) good (0.71) excellent (0.82) good (0.71)
Alignments (obtained from PredictProtein.org)    There were no alignments for this protein in the data base. The BLAST search was initialized and should be ready in a few hours.
Predictions (obtained from PredictProtein.org)
There were no predictions for this protein in the data base. The prediction was initialized and should be ready in a few hours.
The BioBrick-AutoAnnotator was created by TU-Munich 2013 iGEM team. For more information please see the documentation. If you have any questions, comments or suggestions, please leave us a comment.