Part:BBa_K5049006

P_GTH1-Xylanase-GCW61

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.

CONSTRUCTION

The basic parts were built from DNA elements on the pUCIDT-KAN vector of IDT to the iGEM part registry standard pSB1C3 vector, and, in basic parts, designated GTH1 as a registry number of BBa_K5049000, GS-GCW61 as BBa_K5049001 and Xylanase as BBa_K5049003.

The Xylanase-GCW61 fusion protein was connected using the Freiburg assembly method (RFC25)15 to bypass the stop codon of TAG generated by SpeI-XbaI BioBrick scar, developed by the Albert-Ludwigs Universität Freiburg iGEM team in 2007. The composite part was assembled firstly as a registry name of Xylanase-GCW61 and the number of BBa_K5049004. Then, the final composite part was constructed with GTH1 promoter within the context of the following sequence: EcoRI-XbaI-GTH1 promoter-(SpeI/XbaI scar)-Xylanase-(AgeI/NgoMIV scar)-GS linker-GCW61-AgeI-SpeI-PstI. This functional composite part was given an iGEM part registry name of PGTH1-Xylanase-GCW61 and the number of BBa_K5049006.

To express the gene in Pichia pastoris, the composite part was cloned into the pZAHR vector to create the PGTH1-Xylanase-GCW61/pZAHR construct. This construct was verified through colony PCR, using a primer pair targeting the 5’ end of GTH1 and the 3’ end of GCW61, which resulted in an approximately 2100-bp DNA fragment (Figure 1A). Additionally, the integrity of the extracted DNA plasmids was confirmed by digestion with restriction enzymes EcoRI and PstI, yielding DNA fragments of 2070 bp for the insert and 3210 bp for the vector (Figure 1B). Furthermore, the gene sequence of the insert was verified through DNA sequencing performed by Genomics BioSci & Tech. Co. Ltd. in Taiwan.

Figure 1 | Verification of P_GTH1-Xylanase-GCW61/pZAHR construct. (A) Colony PCR using a GTH1-specific forward primer (5’- CCCCAAACATTTGCTCCCCCTAG-3’) and a GCW61-specific reverse primer (5’-AATCAATAGAGCAACACCGGCTA-3’) yielded an expected 2070-bp DNA fragment. The numbers indicate selected colonies, with lane 2 showing a control derived from a mock pick on a clear zone of the agar plate. (B) Plasmids extracted from three successful colony PCR clones underwent a restriction enzyme analysis with EcoRI and PstI. The expected fragment sizes are 2070 bp for the PGTH1-Xylanase-GCW61 insert and 3210 bp for the pZAHR vector. The first lane on the agarose gels features a 1kb DNA marker (FluoroBand™ 1 KB (0.25-10 kb) Fluorescent DNA Ladder, SMOBIO Technology, Inc.).

MEASUREMENT

The transformed Pichia pastoris carrying either the P_GTH1-Xylanase-GCW61/pZAHR construct or the pZAHR vector alone as a control were cultured in BMY media (Buffered Minimal YNB (Yeast Nitrogen Base)). To induce the GTH1 promoter, we added 0.005% (i.e., 0.05 g/L) glucose and incubated the cultures at 28°C for 48 hours to promote xylanase expression. Simultaneously, control groups containing yeast with and without the Xylanase gene were cultured in BMY media without glucose. After incubation, the yeasts were harvested by centrifugation and incubated in the 5% xylan solution at 37°C for 15 minutes to produce xylose. The xylanase activities were measured by the 3,5-dinitrosalicylic acid (DNS) assay. The amount of xylose produced was detected by the color change in the DNS reagent from yellow to reddish-brown at 100°C for 15 minutes, which indicates the presence of reducing sugars. The xylanase activity (Unit/mL/min), is calculated based on the quantity of xylose produced from xylan by the enzyme's catalytic action. For a detailed protocol, including information on prepared buffers and solutions, please refer to our MEASUREMENTpage.

The Xylanase-GCW61 fusion protein was connected using the Freiburg assembly method (RFC25)15 to bypass the stop codon of TAG generated by SpeI-XbaI BioBrick scar, developed by the Albert-Ludwigs Universität Freiburg iGEM team in 2007. The composite part was assembled firstly as a registry name of Xylanase-GCW61 and the number of BBa_K5049004. Then, the final composite part was constructed with GTH1 promoter within the context of the following sequence: EcoRI-XbaI-GTH1 promoter-(SpeI/XbaI scar)-Xylanase-(AgeI/NgoMIV scar)-GS linker-GCW61-AgeI-SpeI-PstI. This functional composite part was given an iGEM part registry name of PGTH1-Xylanase-GCW61 and the number of BBa_K5049006.

To express the gene in Pichia pastoris, the composite part was cloned into the pZAHR vector to create the PGTH1-Xylanase-GCW61/pZAHR construct. This construct was verified through colony PCR, using a primer pair targeting the 5’ end of GTH1 and the 3’ end of GCW61, which resulted in an approximately 2100-bp DNA fragment (Figure 1A). Additionally, the integrity of the extracted DNA plasmids was confirmed by digestion with restriction enzymes EcoRI and PstI, yielding DNA fragments of 2070 bp for the insert and 3210 bp for the vector (Figure 1B). Furthermore, the gene sequence of the insert was verified through DNA sequencing performed by Genomics BioSci & Tech. Co. Ltd. in Taiwan.

Figure 1 | Verification of P_GTH1-Xylanase-GCW61/pZAHR construct. (A) Colony PCR using a GTH1-specific forward primer (5’- CCCCAAACATTTGCTCCCCCTAG-3’) and a GCW61-specific reverse primer (5’-AATCAATAGAGCAACACCGGCTA-3’) yielded an expected 2070-bp DNA fragment. The numbers indicate selected colonies, with lane 2 showing a control derived from a mock pick on a clear zone of the agar plate. (B) Plasmids extracted from three successful colony PCR clones underwent a restriction enzyme analysis with EcoRI and PstI. The expected fragment sizes are 2070 bp for the PGTH1-Xylanase-GCW61 insert and 3210 bp for the pZAHR vector. The first lane on the agarose gels features a 1kb DNA marker (FluoroBand™ 1 KB (0.25-10 kb) Fluorescent DNA Ladder, SMOBIO Technology, Inc.).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 493
  Illegal BamHI site found at 1504
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1831
  Illegal AgeI site found at 1819
  Illegal AgeI site found at 2014
  
• 1000
  COMPATIBLE WITH RFC[1000]