Plasmid construction
The PGIP (PvPGIP1 and PvPGIP2) and PG (AnPG2, BcPG1, BcPG2, and FmPG3)
genes were ordered from Twist Bioscience and codon optimized forS. cerevisiae. These pTwist genes were cloned using the Gateway
system, with a destination vector containing either an AD domain for the
PGIPs or a BD domain for the PGs. The LR Reaction was done using the
guidelines and instructions found in the Invitrogen Gateway LR Clonase
II Enzyme Mix product sheet (Fischer Scientific, catalog #11789020).
Truncated versions of the PGIPs were made using various primers as seen
in Table 1 that ensured the protein would not be spliced in the middle
of the β-sheets. This was checked using the protein 3D imaging software,
PyMOL. Primers were made for the N and C terminus, as well as for each
LRR region to create a multitude of possible PGIP length combinations.
Primers were designed according to the protocol “Gibson Assembly
Cloning” from Addgene. Truncated versions of PGIPs were cloned from the
full length PGIPs using these primers and underwent Gibson assembly with
a vector containing the AD domain.