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Team:Grinnell CN/Project/DspB
From 2011.igem.org
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Latest revision as of 04:13, 27 September 2011
DspB
DspB has been isolated and purified and has been shown to increase dissemination of wild type cells from biofilms and restore dissemination in mutant cells (Kaplan 2003).
DspB comes from the bacteria Aggregatibacter actinomycetemcomitans. It is most likely an N-acetylglucosaminidase that causes the detachment of cells from biofilm colonies through the 1→ 4 glycosidic bond of β-substituted N-acetylglucosamine (Kaplan 2003). DspB is homologous to family 20 glycosyl hydrolases which cleave terminal monosaccharide residues (Kaplan 2003).
One possible substrate for DspB is a type IV pilus required for the attachment and autoaggregation of bacteria by altering its adhesive properties. Another possible substrate is exopolymeric substance (EPS) which is part of the exopolysaccharide matrix which plays a role in biofilm formation. Exopolysaccharides may contain N-acetylglucosamine which DspB is thought to hydrolyze. Manuel et. al have characterized the mechanistic aspects of DspB and show that conserved D183 and E184 resemble the proposed 20 β-hexosaminidase mechanism; that aromatic residues W237, Y278 and D183 assist N-acetal group orientation and hydrolysis; that Y187 plays a role in specificity; and that residues R27, E332 and W330 help stabilize the transition state of hydrolysis (Manuel 2007).
We will test two versions of the gene. The WT gene was sent to us by the University of British Columbia 2010 iGEM Team and was already cloned into pSB1C3. The second is a snthetic version designed with codons optimized for Caulobacter but resulting in the same amino acid sequence. This gene was synthesized by [http://www.idtdna.com/Home/Home.aspx IDT]. BioBrick part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K531004 BBa_K531004].
