RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
MutatedGene model (rodent): PBANKA_1241900; Gene model (P.falciparum): PF3D7_0527100; Gene product: ubiquitin-conjugating enzyme E2 13, putative (UBC13)
Details mutation: the phosphorylating serine 106 replaced with with non-phosphorylatable alanine
PhenotypeNo phenotype has been described
Last modified: 26 May 2022, 18:01
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene mutation
Number of attempts to introduce the genetic modification Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35490588
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherNarwal SK, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute

  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1241900
Gene Model P. falciparum ortholog PF3D7_0527100
Gene productubiquitin-conjugating enzyme E2 13, putative
Gene product: Alternative nameUBC13
Details of the genetic modification
Short description of the mutationthe phosphorylating serine 106 replaced with with non-phosphorylatable alanine
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
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