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| Details of the target gene |
| Gene Model of Rodent Parasite |
PBANKA_1241900
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| Gene Model P. falciparum ortholog |
PF3D7_0527100
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| Gene product | ubiquitin-conjugating enzyme E2 13, putative |
| Gene product: Alternative name | UBC13 |
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| Details of the genetic modification |
| Short description of the mutation | the phosphorylating serine 106 replaced with with non-phosphorylatable alanine |
| Inducable system used | No |
| Short description of the conditional mutagenesis | Not available |
| Additional remarks inducable system |
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| Type of plasmid/construct | (Linear) PCR construct double cross-over |
| PlasmoGEM (Sanger) construct/vector used | No |
| Modified PlasmoGEM construct/vector used | No
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| Plasmid/construct map |
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| Plasmid/construct sequence |
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| Restriction sites to linearize plasmid |
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| Selectable marker used to select the mutant parasite | hdhfr |
| Promoter of the selectable marker | eef1a |
| Selection (positive) procedure | pyrimethamine |
| Selection (negative) procedure | No |
| Additional remarks genetic modification | The unsuccessful attempts to mutate the SER106 residue indicate that Ubc13 phosphorylation at position S106 is essential for parasite viability.
The process of ubiquitylation typically requires three enzymes: E1 (ubiquitin-activating enzyme, Uba), E2 (ubiquitin-conjugating enzyme, Ubc), and E3 (ubiquitin-protein ligase). Amongst Ubcs, Ubc13 specifically catalyzes the formation of K63-linked ubiquitin chains that are critical to signaling in inflammatory and DNA damage response pathways.
Mutations for Ubc13 Ser106 residues were introduced using the NEBQ5 Site-directed Mutagenesis kit (New England Biolabs, USA). To introduce the S/* (A,T) or S/S mutation, the primers were designed as per the manufacturer’s instructions. Primer sets 1517/1183, 1518/1183 and 1180/1183 were used to insert serine/alanine, serine/threonine and serine/serine mutations. Mutations at upstream and downstream of serine 106 was created by using primers 1856/1857. The mutagenic PCR was performed into pBC-Ubc13-mCherry-hDHFR plasmid. The PCR products were digested with DpnI for 2 h at 37 °C and were then transformed into E. coli DH5α cells. The change in the nucleotide was confirmed by sequencing. The mutant plasmids pBC-Ubc13-mCherry-hDHFR_S/* (A/T or S) were linearized |
| Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences  Primer information: Primers used for amplification of the target sequences 
| Sequence Primer 1 | |
| Additional information primer 1 | |
| Sequence Primer 2 | |
| Additional information primer 2 | |
| Sequence Primer 3 | |
| Additional information primer 3 | |
| Sequence Primer 4 | |
| Additional information primer 4 | |
| Sequence Primer 5 | |
| Additional information primer 5 | |
| Sequence Primer 6 | |
| Additional information primer 6 | |
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*RMgm-5208