Difference between revisions of "Part:BBa K5241008"

Latest revision as of 05:25, 30 September 2024

pPICZaAPkrhdd-6xH

Short Description:"pPICZ α" refers to a vector containing the yeast alpha-factor signal peptide sequence, which aids in the proper secretion of proteins. It was developed by Invitrogen (now part of Thermo Fisher Scientific). "pPICZαA-PHDD-6xH" is a vector used for expressing the PHDD protein with a 6xHis tag in Pichia pastoris.

Gene Circuit Diagram:

Description:

Choose the appropriate restriction enzymes to perform double digestion on the pPICZaA plasmid, generating sticky ends that match the target gene fragment (Pkrhdd) and the 6xHis tag sequence. Separate and purify the digested plasmid backbone. Perform a ligation reaction with the purified target gene fragment (Pkrhdd), the 6xHis tag sequence, and the digested pPICZaA plasmid backbone.

Result:

We are using chemical and electroporation methods to transform the pPICZaAPkrhdd-6xH into Pichia pastoris, and colonies have formed after cultivation, but there is no melanin expression after induction with methanol. This could be due to a failure in plasmid construction, or it could be due to other reasons.

Reference documentation

[1] Gätjen, D., Tomszak, F., Dettmann, J. C., Droste, M., Nölle, V., & Wieczorek, M. (2022). Design of a novel switchable antibody display system in Pichia pastoris. Applied Microbiology and Biotechnology, 106(18), 6209-6224. https://doi.org/10.1007/s00253-022-12108-5

Sequence and Features