Xylan defines a group of hemicelluloses, polysaccharides based on xylose, mainly occurring in plant cell walls. In hardwoods up to 35 % of the whole hemicellulose content are Xylanes (1). Also the Xylose Monomer is suitable for generation of photopolymers and therefore of use in our project. For xylan degradation three enzymes were chosen, which cleave either the xylan main chain (xynA (2)) or side chains (aes(3), Ruxyn1). The side chains of xylan vary by the type of utilized xylan. Xylan of hardwood mainly has acetyl groups, while softwood xylan lacks acetyl groups but has arabinose residues instead. Both xylan types are targeted by aes (degradation of acetyl residues) and Ruxyn1 (degradation of arabinose residues).Luckily all three degradation enzymes were already listed in the iGEM registry and ready for usage in our model system. The parts we used were BBa_K805012, BBa_K1216002, BBa_K1175005.
In this model degradataion pathway the protein scaffold by iGEM TU Darmstadt 2014 (based on Dueber et al., 2009 (7)) was fused with a 15 amino acid long peptide (silica-tag) (6), binding to silica surfaces as shown by iGEM Leeds 2013. Green Fluorescent Protein (GFP) was fused with the silica-tag for proving silica-binding abilities of the peptide. For easy purification of both fusion proteins N-terminal polyhistidine tags were added. For increasing the degradation efficiency of xylan, protein scaffolding was prepared by fusing the cognate binding peptides of the three protein domains GBD, SH3 and PDZ to the degradation enzymes Ruxyn1, aes and xynA, respectively. All coding sequences were assembled in an operon under the control of a T7 promoter system, which was co-transformed with the scaffolding plasmid into E. coli Top10. In the next step three different characterization elements for later on optimization are important:
1) To check the binding to a silica-surface and stability of boundary we fused the Si4-tag by iGEM Leeds 2013 (2) with GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) using Overlap PCR / Biobrick Assembly. After Expression and Purification we want to visualize the boundary to a silica-coated surface in different buffer conditions and time dependency off the boundary.
2) By linking the zinc finger ligand sequence to GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) and combining purified protein scaffold domains with the fluorescent proteins we want to control the boundary of ligand scaffold domain in different buffer conditions.
3) To verify the advantage of our protein scaffolding we want to compare the yield of degrading xylan only based on the three degradation enzymes in one composite to the combination of the composite and the protein-scaffold.
Exemplarily the overexpression of degradation enzyme Acetyl Esterase fused to a linker binding the SH3 domain of the protein scaffold is shown.
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