IGEM Bordeaux 2015


Our submitted genes are listed down below


Curdlan synthase gene CrdS with codon optimization for E. coli : BBa_K1686046

Curdlan is a bacterial polysaccharide (linear (1->3)-β-glucan) that has been of significant recent interest due to its valuable properties and its bioactivity. In fact, 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 (1->3)-β-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.

CrdS under the control of a stationary phase promoter (pOsmY) : BBa_K1686033

BBa_K1686033 is a composite part consisting of an Escherichia coli osmY stationary phase promoter (BBa_J45992) and Curdlan synthase gene (BBa_K1686046). Thus, BBa_K1686033 produces curdlan synthase in stationary phase cultures.

See BBa_J45992 for details on the osmY stationary phase promoter.
See BBa_K1686046 for details on the Curdlan synthase gene.

Full-length stationary phase osmY promoter (MIT 2006 iGEM team) : BBa_J45992

BBa_J45992 was no longer in stock so we submitted it once again. We used it to express our Curdlan synthase gene (crdS) BBa_K1686046 in stationary phase. BBa_J45992 worked as expected, activating our gene only in stationary phase.

crdA gene with codon optimisation for E. coli : BBa_K1686045

CrdA might assist translocation of the nascent polymer of Curdlan across the cytoplasmic membrane.
Note : This protein has not really well described in literature. However this gene is essential for curdlan production and may increase curdlan production in our chassis.

crdC gene with codon optimisation for E. coli : BBa_K1686047

CrdC assists translocation of the nascent polymer across across the periplasm.
Note : This protein has not really well described in literature. However this gene is essential for curdlan production and may increase curdlan production in our chassis.

His-Gal promoter BBa_K1686098

BBa_K1686098 is a biobrick containing a histidine auxotrophy gene of yeast and the promoter galactose one.
Note : The sequence submitted in the part sequence is the initial sequence of the promoter. We have to do a mutagenesis to delete the different sites of restrictions that are not allowed in the final sequence. Our team had not the time to do it, that's why we only gave us the initial sequence in pSB1C3.

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