Translational_Unit

Part:BBa_K4317117

Designed by: Chris Song   Group: iGEM22_Seoul_Korea   (2022-10-12)


KsCel7A-T2A-sfGFP

Our team used two previously registered parts to facilitate the screening process. One is T2A (BBa_K1993019), a self-cleaving peptide, and the other is sfGFP (BBa_I746916). In order to obtain a transformant that effectively expresses and secretes Kscel7A, known as exo-cellobisidase I, in Pichia pastoris, T2A and sfGFP were fused to the back of the KsCel7A coding sequence with the stop codon removed (Our new composite parts : BBa_K4317116; T2A-sfGFP, BBa_K4317117; KsCel7A-T2A-sfGFP). In the figure 1, the recombinant plasmid DNA was transformed into Pichia pastoris through electroporation. Colonies selected in the zeocin-added medium were patched on an MM agar plate containing methanol. After incubation at 30°C for 2 days, use a fluorescent device Figure 2) to find transformants expressing sfGFP (Figure 3). Our new composite parts and methods make it easy to find transformants expressing the target protein in pichia. 577px-PPICZalpha-KsCel7A-T2A-sfGFP.png

Figure 1. Plasmid map for expression of KsCel7A fused with T2A-sfGFP


Usage and Biology

800px-Blueled.jpg

Figure 2. Blue LED and plastic emission filter for GFP detection

Pichia_t2a.jpg

Figure 3. Pichia transformants expressing sfGFP.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1593
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1665


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