Team:Toulouse/Project/binding

The second step in the SubtiTree optimization matches with the binding ability of our bacterium. Thus, we design a chimeric protein (BBa_K1364005) able to make a bridge between bacterial peptidoglycan and fungal chitin , the main component of the pathogen’s cell wall. According to the Imperial College of London 2010 iGEM team, we use CWB domain of LytC protein to bind our chimeric protein to Bacillus subtilis cell wall. On the other side of our protein, we add the fragment of GbpA from Vibrio Cholerae , which is known to recognize N-Acetyl Glucosamine oligosaccharides called chitin.

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.

To introduce the Binding Module in Bacillus subtilis chromosome, we insert the ORF Binding Module in pSBBS4S plasmid (BBa_K823022), from the LMU-Munich 2012 iGEM team, with:
- Regulatory section : after the ORF Binding Module, we include Pveg (BBa_K143012) as strong promoter and iGEM RBS consensus (BBa_K090505).
- Transcription end : we use a double terminator (BBa_B0015)

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