Difference between revisions of "Part:BBa K1686046"
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===Usage and Biology===
 
===Usage and Biology===
 
Curdlan is a bacterial polysaccharide that has been of significant recent interest due to its interesting and valuable properties and its bioactivity.  
 
Curdlan is a bacterial polysaccharide that has been of significant recent interest due to its interesting and valuable properties and its bioactivity.  
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Curdlan has been used traditionally in food products for calorie-reduced food (freezable tofu noodles) and its gel properties. Moreover, Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses. For example, it can have antitumor, anti-infective, anti-inflammatory, and anticoagulant activities (see other properties of Curdlan). In particular, this β1,3 glucan can stimulate plants' immune system.
  
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In fact crdS codes Curdlan Synthase.
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Our project is focused on the production of curdlan for stimulate plant defense system against Downy Mildew.  
  
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The crdS product (73 kDa) deduced from the DNA sequence (1,965 bp) is curdlan synthase. In Agrobacterium, CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition
 
  
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The molecular genetics of curdlan production have been investigated in Agrobacterium sp. ATCC31749. Studies on transposon-insertion mutants with altered capacity to stain with aniline blue when grown on indicator agar identified four genes (crdA, crdS, crdC, crdR) that are essential for curdlan production
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The molecular genetics of curdlan production have been investigated in Agrobacterium sp. ATCC31749.
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Studies identified three genes (crdA, crdS and crdC) that are essential for curdlan production
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The crdASC genes occupy a contiguous 4,948 bp region of the genome ( the crdS gene is flanked by the crdA and crdC genes).
  
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The crdS gene (1965 bp) encodes the curdlan synthase (CrdS, 73 kDa) protein.
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In Agrobacterium, CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition.
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The UDP-Glucose substrate-binding and the catalytic motif for curdlan polymerisation is observed between TM3 and TM4.
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The process of curdlan polymerisation mediated by CrdS occurs on the cytoplasmic face of the inner membrane
  
  
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For our project, we wanted to produce curdlan by E.coli, so this part has been made with a codon optimization of the natural crdS sequence for E.coli.
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A central large and relatively hydrophilic cytoplasmic re- gion of !300 residues situated between TM3 and TM4 carries various conserved motifs, including the UDPGlc substrate-binding and catalytic D,D,D35QxxRW motif
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The crdS gene of Agrobacterium sp. strain ATCC31749 encodes the curdlan synthase (CrdS) protein
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encodes a membrane protein
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Locus I is a putative operon of three genes in which the crdS gene, encoding a protein homologous with family GT- 2 glycosyl transferases (Coutinho and Henrissat, 1999), is flanked by the crdA and crdC genes (Stasinopoulos et al., 1999). The latter two genes encode proteins sharing no significant homology with reported protein sequences, and their roles in curdlan production are unknown.
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Revision as of 13:26, 12 September 2015

Curdlan synthase gene with codon optimisation for E. coli


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 678
    Illegal XhoI site found at 400
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 801
    Illegal AgeI site found at 1192
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Curdlan is a bacterial polysaccharide that has been of significant recent interest due to its interesting and valuable properties and its bioactivity. Curdlan has been used traditionally in food products for calorie-reduced food (freezable tofu noodles) and its gel properties. Moreover, Curdlan belongs to the class of biological response modifiers that enhance or restore normal immune defenses. For example, it can have antitumor, anti-infective, anti-inflammatory, and anticoagulant activities (see other properties of Curdlan). In particular, this β1,3 glucan can stimulate plants' immune system.

Our project is focused on the production of curdlan for stimulate plant defense system against Downy Mildew.


The molecular genetics of curdlan production have been investigated in Agrobacterium sp. ATCC31749. Studies identified three genes (crdA, crdS and crdC) that are essential for curdlan production The crdASC genes occupy a contiguous 4,948 bp region of the genome ( the crdS gene is flanked by the crdA and crdC genes).

The crdS gene (1965 bp) encodes the curdlan synthase (CrdS, 73 kDa) protein. In Agrobacterium, CrdS is an integral inner membrane protein with seven transmembrane (TM) helices, one non-membrane-spanning amphipathic helix and a Nout–Cin disposition. The UDP-Glucose substrate-binding and the catalytic motif for curdlan polymerisation is observed between TM3 and TM4. The process of curdlan polymerisation mediated by CrdS occurs on the cytoplasmic face of the inner membrane


For our project, we wanted to produce curdlan by E.coli, so this part has been made with a codon optimization of the natural crdS sequence for E.coli.