title: An integrated strategy for efficient vector construction and multi-gene expression in Plasmodium falciparum.
abstract: BACKGROUND
  The construction of plasmid vectors for transgene expression in the malaria parasite, Plasmodium falciparum, presents major technical hurdles. Traditional molecular cloning by restriction and ligation often yields deletions and re-arrangements when assembling low-complexity (A + T)-rich parasite DNA. Furthermore, the use of large 5'- and 3'- untranslated regions of DNA sequence (UTRs) to drive transgene transcription limits the number of expression cassettes that can be incorporated into plasmid vectors.
  
  
  METHODS
  To address these challenges, two high fidelity cloning strategies, namely yeast homologous recombination and the Gibson assembly method, were evaluated for constructing P. falciparum vectors. Additionally, some general rules for reliably using the viral 2A-like peptide to express multiple proteins from a single expression cassette while preserving their proper trafficking to various subcellular compartments were assessed.
  
  
  RESULTS
  Yeast homologous recombination and Gibson assembly were found to be effective strategies for successfully constructing P. falciparum plasmid vectors. Using these cloning methods, a validated family of expression vectors that provide a flexible starting point for user-specific applications was created. These vectors are also compatible with traditional cloning by restriction and ligation, and contain useful combinations of commonly used features for enhancing plasmid segregation and site-specific integration in P. falciparum. Additionally, application of a 2A-like peptide for the synthesis of multiple proteins from a single expression cassette, and some rules for combinatorially directing proteins to discrete subcellular compartments were established.
  
  
  CONCLUSIONS
  A set of freely available, sequence-verified and functionally validated parts that offer greater flexibility for constructing P. falciparum vectors having expanded expression capacity is provided.
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Wagner
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Goldfless
authors.forename: Suresh M
authors.initials: SM
authors.lastname: Ganesan
authors.forename: Marcus C S
authors.initials: MC
authors.lastname: Lee
authors.forename: David A
authors.initials: DA
authors.lastname: Fidock
authors.affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. jcniles@mit.edu.
authors.forename: Jacquin C
authors.initials: JC
authors.lastname: Niles
mesh_terms: D015870:Gene Expression; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D010963:Plasmodium falciparum / Q000235:genetics*; D019076:Transgenes