Summary

RMgm-4412
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1121900; Gene model (P.falciparum): PF3D7_0623000; Gene product: chorismate synthase (CS)
PhenotypeNo phenotype has been described
Last modified: 2 February 2018, 16:24
  *RMgm-4412
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 29338985
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei TRAP/FlpL
Other information parent lineThis line expresses FlpL recombinase under the control of the TRAP promoter, active in the mosquito oocyst stage, and selectively excises the DNA sequence flanked by FRT sites (S. Mishra, K.A. Kumar and P. Sinnis, unpublished data). The FlpL expression cassette is introduced into the silent p230p gene locus (by GIMO transfection using line 1596cl1; RMgm-687) resulting in line 1809. The line has been generated in Leiden and cloned in the Sinnis lab. This line does not contain a drug-selectable marker.
The mutant parasite was generated by
Name PI/ResearcherChoudhary HH, Mishra S
Name Group/DepartmentDivision of Parasitology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-4412
Principal nameCs mutant
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of Chorismate synthase (Cs). The mutant was obtained after excision of the cs gene by the Flp/FRT system. The mutant expresses Flp recombinase under the control of the promoter of trap. The cs gene has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the cs gene has been achieved by  transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the cs gene which was flanked by FRT sequences. After mosquito passage parasites were cloned and cloned mutants were selected and analysed that lack the cs gene

Protein (function)
The shikimate pathway has seven steps that finally  lead to the formation of chorismate from 5-enolpyruvylshikimate 3-phosphate (EPSP). Chorismate produced as the end product of this pathway serves as a substrate for the synthesis of aromatic amino acids, Para-aminobenzoic acid (PABA) and ubiquinones. PABA is a precursor for folates that are required for nucleotide synthesis and the methylation cycle. Ubiquinones are components of the electron transport chain, and in Plasmodium it is synthesised in two steps – the isoprene side chain synthesis occurs via a terpenoid pathway, and benzoquinone synthesis occurs via a shikimate pathway. Chorismate synthase (Cs) is the immediate and only enzyme of this pathway required for chorismate synthesis. CS carries out 1, 4 trans elimination of the phosphate group of EPSP that results in the formation of chorismate, utilising Flavin mononucleotide (FMN) as a cofactor.

Phenotype
No phenotypes were detected in blood, mosquito and liver stages that were different from wild type.

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1121900
Gene Model P. falciparum ortholog PF3D7_0623000
Gene productchorismate synthase
Gene product: Alternative nameCS
Details of the genetic modification
Inducable system usedFlp/FRT
Additional remarks inducable system The mutant lacks expression of Chorismate synthase (Cs). The mutant was obtained after excision of the cs gene by the Flp/FRT system. The mutant expresses Flp recombinase under the control of the promoter of trap. The cs gene has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the cs gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the cs gene which was flanked by FRT sequences. After mosquito passage parasites were cloned and cloned mutants were selected that lack the cs gene
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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