Difference between revisions of "Part:BBa K5049006"

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     In 2016, iGEM Team TJUSLS-China made significant progress in developing PET-degrading enzymes, specifically PETase from <em>Ideonella sakaiensis</em> 201-F6. They anchored PETase to the surface of <em>Pichia pastoris</em> using the GPI-related cell wall protein GCW61. Inspired by their work, we explored whether adding anchor proteins enhances PETase functionality and identified potentially better anchors, such as Pir1 from <em>Saccharomyces cerevisiae</em>. We conducted 3D protein structure modeling to compare wild-type PETase, PETase-GCW61, and PETase-Pir1 in terms of 3D images, ligand binding site residues, active site residues, and stability (free energy). These analyses determined the benefits of using anchor proteins and suggested potential improvements in anchor selection.
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     The sequences of DNA elements for the GTH1 promoter and GCW61 anchor protein were from Pichia pastoris. To ensure flexibility in connecting to target fusion proteins, a GS linker ((GGSG)3) was added at the N-terminus of GCW61. We selected a thermostable xylanase from Streptomyces thermovulgaris6 as our protein of interest. All DNA fragments were synthesized by Integrated DNA Technologies (IDT) following the standard iGEM Part Registry Rule (RFC10)14, which includes prefix cutting sites EcoRI and XbaI, and suffix cutting sites SpeI and PstI. In the issue of the assembly of the fusion protein, we followed the rules created by the Albert-Ludwigs Universität Freiburg iGEM team in 2007 (Freiburg assembly method, officially named by iGEM HQs as RFC25)15. AgeI cutting site was introduced at C-terminus of the Xylanase gene without the stop codon, and NgoMIV and AgeI sites were introduced at the either end of the GS linker-GCW61 segment.  
 
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To create the vector for gene expression in P. pastoris, we utilized the yeast vector pZAHR, developed by Professor Hung-Jen Liu's lab at National Chung Hsing University. This vector is a Zeocin-selectable, AOX1-based Homologous Recombination vector designed specifically for gene knock-in applications in Pichia pastoris. It incorporates the AOX1 gene promoter and terminator to facilitate the integration of desired genes into the Pichia pastoris chromosome through homologous recombination. This process is typically executed following electroporation-directed yeast transformation, a method routinely employed in the Liu’s laboratory.
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Revision as of 08:22, 2 September 2024


PGTH1-Xylanase-GCW61

VECTOR DESIGN

Vector Design

The sequences of DNA elements for the GTH1 promoter and GCW61 anchor protein were from Pichia pastoris. To ensure flexibility in connecting to target fusion proteins, a GS linker ((GGSG)3) was added at the N-terminus of GCW61. We selected a thermostable xylanase from Streptomyces thermovulgaris6 as our protein of interest. All DNA fragments were synthesized by Integrated DNA Technologies (IDT) following the standard iGEM Part Registry Rule (RFC10)14, which includes prefix cutting sites EcoRI and XbaI, and suffix cutting sites SpeI and PstI. In the issue of the assembly of the fusion protein, we followed the rules created by the Albert-Ludwigs Universität Freiburg iGEM team in 2007 (Freiburg assembly method, officially named by iGEM HQs as RFC25)15. AgeI cutting site was introduced at C-terminus of the Xylanase gene without the stop codon, and NgoMIV and AgeI sites were introduced at the either end of the GS linker-GCW61 segment.

To create the vector for gene expression in P. pastoris, we utilized the yeast vector pZAHR, developed by Professor Hung-Jen Liu's lab at National Chung Hsing University. This vector is a Zeocin-selectable, AOX1-based Homologous Recombination vector designed specifically for gene knock-in applications in Pichia pastoris. It incorporates the AOX1 gene promoter and terminator to facilitate the integration of desired genes into the Pichia pastoris chromosome through homologous recombination. This process is typically executed following electroporation-directed yeast transformation, a method routinely employed in the Liu’s laboratory.


Vector Design

===Sequence and Features=== BBa_K5049006 SequenceAndFeatures