Team:Toulouse/Project/binding

In order to be more effective in the fight against the pythopathogen, our optimized bacterium has to be anchored to the fungus. This module matches with the binding ability of SubtiTree. Thus, we designed a chimeric protein (BBa_K1364005) to make a bridge between the bacterial peptidoglycan and the fungal chitin , which is the main component of the pathogen’s cell wall. According to the work of the Imperial College 2010 iGEM team, we used the CWB domain of the LytC protein (coding for a N-acetylmuramoyl-Lalanine amidase) to bind our chimeric protein to Bacillus subtilis cell wall. On the other side of our protein, we added the domain 4 of GbpA from Vibrio Cholerae , which is known to recognize N-AcetylGlucosamine oligosaccharides called chitin.

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The Binding Module ORF is composed of 3 sections:

• Anchor section : the CWB (Cell Wall Binding) domain is extracted from LytC gene and composed the 5' side of our binding module. As previously used by the Imperial College of London 2010 iGEM team, we kept the first 318 bp. We can note the presence of the signal peptide at the beginning of the sequence, from 1 to 24bp.

• Chitin Binding Domain (CBD) section : the Domain 4 of GbpA from Vibrio Cholerae is able to bind to N-Acetyl Glucosamine oligosacchararides. Also, the 3' side 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 used the same six amino acids sequence (SRGSRA) to make a bridge between the Anchor section and the Chitin Binding section.

The sequence has been designed in silico and codon optimized for the transcription in B. subtilis.

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The binding module has been placed under the control of Pveg (BBa_K143012), a strong constitutive promoter and we used a consensus RBS (BBa_K090505) and a double terminator (B0015). The construct has been inserted in an integrative plasmid, pSBBS4S (BBa_K823022)which comes from the LMU-Munich 2012 iGEM team.

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).

Chemotaxis

Fungicides