Part:BBa_K5049007

P_GTH1-Xylanase-Pir1

CONSTRUCTION

Pir1 is another anchor protein of Saccharomyces cerevisiae, widely utilized in yeast display systems. To explore its potential compared to GCW61, we aimed to construct a Xylanase-Pir1 fusion protein. Using the same gene expression strategy as with GCW61, the Pir1 sequence from Saccharomyces cerevisiae was synthesized with a GS linker at the N-terminus, flanked by EcoRI-XbaI-NgoMIV as a prefix and AgeI-SpeI-PstI as a suffix by IDT. This basic part, GS-Pir1, was assembled into the pSB1C3 vector (BBa_AAAA), and the composite part, PGTH1-Xylanase-Pir1/pSB1C3, was then created (BBa_BBBB). To evaluate its performance in yeast and compare it with the GCW61 anchor, the PGTH1-Xylanase-Pir1 construct was cloned into the pZAHR vector. The construct was verified by colony PCR (Figure 3) and confirmed by DNA sequencing.

Figure 1 | Verification of PGTH1-Xylanase-Pir1/pZAHR construct. Colony PCR using a GTH1-specific forward primer (5’- CCCCAAACATTTGCTCCCCCTAG-3’) and a Pir1-specific reverse primer (5’-AGAAGTTAAAGTTGTGGCTTG-3’) yielded an expected 2346-bp DNA fragment. The numbers indicate selected colonies, with lane 1 showing a 1kb DNA marker on 1% agarose gel in 0.5x TAE buffer (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 2344
  
• 1000
  COMPATIBLE WITH RFC[1000]