Additional remarks phenotype | Mutant/mutation
The mutant expresses GFP under control of the bip promoter
Protein (function)
Phenotype
In order to create a compact, constitutive promoter and to monitor its transcriptional strength in a population of parasites, one of two short (~ 300 bp, ~ 500 bp) versions of the pybip promoter were fused upstream of GFPmut2, as was previously done for the pbeef1a promoter. Asexual blood stage parasites were synchronized to schizonts in an ex vivo culture to reduce differences in protein levels that could be attributable to the smaller ring and trophozoite stage parasites. As seen in biological duplicate by flow cytometry assays, the control pbeef1a promoter and 500 bp pybip promoter constructs allowed transcription above background fluorescence of untransfected Py17XNL parasites, while the 300 bp pybip promoter construct did not. Transcription from the pbeef1a promoter produced a broader and stronger expression distribution, including two discernable populations of high/highest expression. Similarly, the 500 bp version of the pybip promoter allowed robust transcription with two discernable peaks of gene expression. While no appreciable expression was detected by flow cytometry for the 300 bp pybip promoter, a few cells with GFP above background levels were detected by live fluorescence microscopy. Together this indicates that cis elements critical for robust transcription reside in the − 500 to − 300 region of this promoter, and that a compact and strong pybip promoter can be defined.
Additional information
From the literature, promoters that have been described to be strong and constitutive or stage specific as observed in other Plasmodium species were selected for study. To produce as diverse of a promoter collection as possible, individual promoters with expression profiles that span the complete life cycle of Plasmodium parasites were chosen. The clag-a promoter (PY17X_1402200) has been defined as restricting transcription to the asexual blood stage of the related rodent malaria parasite P. berghei. The dynein heavy chain delta (“dd”, male, PY17X_0418900) and lap40 (also called ccp2, female, PY17X_1323300) promoters have been commonly used in the “820” male/female fluorescent protein reporter line (820cl1m1cl1). The trap promoter (PY17X_1354800) is known to be active throughout sporozoite development and is critical for salivary gland invasion, gliding motility, and infectivity, whereas the uis4 promoter (PY17X_0502200) flips from being weakly active in oocyst sporozoites to becoming one of the strongest promoters in salivary gland sporozoites. Finally, the lisp2 promoter (PY17X_1004400) becomes active in midliver stage and remains so throughout liver stage development [34]. Finally, the bip promoter (PY17X_0822200) was also selected, as it is known to be a strong and constitutive promoter in eukaryotes due to the integral role that the BiP protein plays in the translocation of nascent, unfolded proteins into the ER. In previous work, a 1.5 kb portion of sequence upstream of the bip coding sequence was used to serve as a promoter for CRISPRbased gene editing. Here, two truncated variants were created in an attempt to produce a strong, minimal constitutive promoter. Promoters tested here typically included 1.6–1.8 kb of sequence upstream of (and including) the start codon, and were designed to place a unique restriction site 3′ of the start codon to avoid effects upon translation that may occur if placed upstream of it.
Other mutants |