Part:BBa_K1364005

Pveg + Chitin Binding Protein for Bacillus subtilis

Binding chimeric protein (BBa_K1364005) is able to make a bridge between bacterial peptidoglycan and fungal chitin , the main component of the pathogen’s cell wall.

Sequence and Features

The Binding Module ORF is composed of 3 sections: - Anchor section : the CWB (Cell Wall Binding) is a LytC domain put on 5' of our chimeric protein gene. As previously used by the Imperial College of London 2010 iGEM team, we retain the first 318 bp. We can note the presence of the signal peptide at the beginning from 1 to 24 bp. - Chitin Binding Domain (CBD) section : the Domain 4 of GbpA from Vibrio Cholerae is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the last base pairs in 3' of our gene is composed by a part of the GbpA sequence (from 423 to 484 bp). - Helical Linker : according to the work of the Imperial College of London 2010 iGEM team, we use the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.

Assembly Compatibility:

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

  Illegal BglII site found at 381
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1034
  Illegal AgeI site found at 657
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functionnal characterization of the BioBrick :

The synthetic bacterium,with the binding module, is put with special beads composed of the polymer miming the fungal pathogen wall. After several washes, bacteria specifically attached to the chitin are put on plates and counted. 10^5 bacteria per mL are used during thing test. Even though there is no significant difference between both strains after the first wash, the second wash has a major effect since it allows 40 times more Wild Type bacteria to come off the beads. This result correlates with the number of bacteria binded to the beads for the synthetic strain with the binding module. Thus, the binding system seems to function correctly and leads to the bacterial attachment on the chitin.

Through the use of a fluorochrome (Syto9), we can highlight the presence of bacteria on the surface of the beads (confocal laser scanning microscope)

Finally, overall results are consistent with the presence of functional binding system.

More information on our wiki : http://2014.igem.org/Team:Toulouse

References
M. Desvaux, E. Dumas, I. Chafsey and M. Hébraud. Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure . FEMS Microbiol. Lett. 256, 1–15 (2006). E. Wong, G. Vaaje-Kolstad, A. Ghosh, R. Hurtado-Guerrero, PV. Konarev, AF. Ibrahim, DI. Svergun, VG. Eijsink, NS. Chatterjee and DM. van Aalten.The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog. 8, e1002373 (2012). H. Yamamoto, S. Kurosawa and J. Sekiguchi. Localization of the vegetative cell wall hydrolases LytC, LytE, and LytF on the Bacillus subtilis cell surface and stability of these enzymes to cell wall-bound or extracellular proteases. J. Bacteriol. 185, 6666–6677 (2003).