"
Team:Toulouse/Project/binding
From 2014.igem.org
| Line 62: | Line 62: | ||
<!--Short description : à changer!!!--> | <!--Short description : à changer!!!--> | ||
| - | <p> 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 | + | <p> The second step in the SubtiTree optimization matches with the <B> binding ability </B> of our bacterium. Thus, we design a chimeric protein (BBa_K1364005) able to make <B> a bridge between bacterial peptidoglycan and fungal chitin </B>, 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.</p> |
<B> More information about this module </B> | <B> More information about this module </B> | ||
| Line 68: | Line 68: | ||
<br>- 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. | <br>- 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. | ||
<br>- 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). | <br>- 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). | ||
| - | <br>- 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. | + | <br>- 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.</p> |
| - | </p> | + | |
<B>Final construction: (see also the section “Parts” of the wiki to have more details about the intermediate parts)</B> | <B>Final construction: (see also the section “Parts” of the wiki to have more details about the intermediate parts)</B> | ||
Revision as of 12:34, 1 October 2014
Binding
Proin sollicitudin nibh ut dapibus vulputate.
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.
More information about this module 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)
Socialize
