Figure 3
From: A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris
(A) Redesigning pPICZαA for PCR product transformation based on amplification with primers αA_PmeI_F (“F”) and αA_PmeI_R (“R”). The pUC ori region was moved from the 3′ terminus of the CYC1 transcription terminator in pPICZαA [Top] to the PmeI site of r_αA [Bottom], splitting the 5′ AOX1 promoter region. A 64 base pair reduction in size is due to removal of a 10 base pair section between CYC1 transcription terminator and the pUC ori, and a further 54 base pairs between the pUC ori and the 5′ AOX1 promoter region. Overall, the size of the PCR amplicon to produce the integration cassette is reduced by 683 bp. (B) TPA activity of culture supernatants from clones integrating pPICZαA-PLAT or r_αA-PLAT following 120 hours of induction. Data represent the average of six clones for each of GS115[αA-PLAT] and GS115[r_αA-PLAT]; for no enzyme, no substrate and GS115 the data are the average of three independent reactions/clones. ANOVA tests were conducted on the data; significant differences are indicated by asterisks where **p < 0.01 and *p < 0.05. Post-hoc Tukey tests revealed activity in GS115 supernatants to be significantly different from GS115[αA-PLAT] (p-value = 0.00621) and from GS115[r_αA-PLAT] (p-value = 0.0475) supernatants.
