Journal: bioRxiv

Article Title: Assessment of biological role and insight into druggability of the Plasmodium falciparum protease plasmepsin V

doi: 10.1101/426486

Figure Lengend Snippet: (A) Schematic of pSN054 showing restriction sites for cloning (dashed lines), loxP sites for DiCre operations (red triangles), and aptamers (black lollipops). 2A (pink) is a viral skip peptide. Restriction sites allow the choice of N- or C-terminal 3xHA (blue), FLAG (green), or Myc (purple). The plasmid also contains an expression cassette driving production of the Tet repressor and DOZI helicase fusion (TetR-DOZI, black), Renilla luciferase (Ren. Luc.), and blasticidin-S deaminase selectable marker (BSD). The T7 expression cassette is for driving transcription of CRISPR guide RNAs (gRNA), which can be inserted by cutting with I-ppoI or AflII (contained within the I-ppoI cut site). (B) Diagram showing the cloning strategy for editing of the PMV locus. Left and right homologous regions (LHR and RHR) were inserted at FseI and I-SceI respectively, while the recoded gene sequence was inserted into plasmid cut with AsiSI and BsiWI. The endogenous sequence of PMV was disrupted by CRISPR/Cas9 gene editing. When transcribed, aptamers are bound by TetR-DOZI in the absence of aTc, repressing translation. In the presence of aTc, TetR-DOZI does not bind the aptamers and translation occurs as normal.

Article Snippet: Transfectants were maintained in 0.5 μM anhydrotetracycline (aTc; Cayman Chemical) and were selected beginning 24 hours post transfection with Blasticidin S (2.5 μg/mL; Fisher).

Techniques: Clone Assay, Plasmid Preparation, Expressing, Luciferase, Marker, CRISPR, Sequencing