Improvement of an Existing Part

Improvement

We used the pPICZ-alpha plasmid for the expression of heterologous proteins in Pichica pastoris. This plasmid enters the plasma membrane of Pichia pastoris, moves to the nucleus, and is integrated into the AOX1 locus in the chromosome. This ensures stable expression of the desired protein. However, in this process, for various reasons, there are transformants that are resistant to antibiotics, but cannot express proteins.

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.4