Using biolistic transformation and a mutant construct based on the work of Cassidy-Hanley et al. [15], we were able to disrupt the macronuclear KIN5 locus.
For overexpression of mutant alleles, new constructs based on the pRS425 vector (Christianson et al. 1992) were created, by subcloning mutated sequences using the SLIC method (Li and Elledge 2007).
Using R-3-chloromandelic acid as a model substrate, a total of 53 mutants were constructed based on rational design in the two rounds of screening.
A series of mutants were constructed based on the two genetically different, but functionally identical helper phage genomes, M13K07 and VCSM13.
Expression vectors for human PTPMeg2 and PTPMeg2C515S or deletion mutants were constructed based on pcDNA3.1-Myc or pEFneo-Myc.
The mutant G276R was constructed based on the structure of wild-type CueO, which was modeled using the Discovery Studio 2.5.5 software.
Additionally, three mutant model systems were constructed based on the native model: a mutant lacking the β-hairpin (Δ233 240) (sim2, hairpin mutant), a mutant lacking the α-helix (Δ447 460) (sim3, helix mutant), and a mutant lacking both (Δ233 240 and Δ447 460) (sim4, helix-hairpin mutant).
Involvement of the C-terminal region of Hsp70 in M1 binding was confirmed; thus, to identify the region of the M1 protein involved in this binding, deletion mutants of M1 were constructed based on a previous report.
The sliding glycosylation mutant construct was obtained based on tyrosinase mutant cDNA Δ7 with the following codon changed: Q378N (s7→378).
The mutant 3′-UTR of PLD1, which carried the mutated sequence in the complementary site for the seed region of miR-638, was constructed based on the p-Luc-PLD1 3′UTR-WT plasmid by overlap-extension PCR.
