PlasmoGEM gene targeting vectors

PlasmoGEM uses a combination of recombineering and Gateway® technology to convert genomic libraries into gene targeting vectors with long homology arms, which integrate with high efficiency (Pfander et al., 2011).

All PlasmoGEM vectors are equipped with sequence-readable barcodes, and we use barcode-sequencing (BarSeq) for high-throughput parallel growth phenotyping of mutants (method in Gomes et al., 2015).

The first step in the vector production pipeline uses recombineering (recombinase-mediated engineering; Zhang et al. 1998, Wang et al., 2006) and produces an intermediate vector in which a Zeo/PheS bacterial resistance marker is inserted into the coding sequence (gene knock-out) or 3'UTR (c-terminal tagging) of the target locus. The process is completed by a Gateway®-mediated exchange of the Zeo/PheS cassette for an hdhfr-yfcu parasite selectable marker.

Two types of gene targeting vectors are currently available from PlasmoGEM:

• Gene knock-out (complete or partial deletion) vectors.
• C-terminal epitope tagging vectors.

Final and intermediate PlasmoGEM gene targeting vectors, as well as toolkit vectors can be requested free of charge:

• Final PlasmoGEM gene targeting vectors are ready to be prepped for P. berghei transfection.
• Intermediate PlasmoGEM c-terminal tagging vectors where the Zeo/PheS cassette can be swapped for a P. berghei 3'-tag of choice. A protocol for doing so is available here.
• Toolkit Gateway vectors with different flavour fluorescent tagging cassettes including mCherry, iLOV, eGFP, eMERALD and mVenus.