Pooled transfections

Each of 2578 knockout vectors was assigned to one or more vector pools, creating a total of 58 pools of vectors. For each pooled transfection, vectors were prepared and transfected largely as described (Gomes et al., 2015) with minor modifications. Briefly, groups of 96 targeting vectors were prepared in parallel by growing 1 mL liquid cultures inoculated from glycerol stocks into duplicate 96X deep-well plates, incubated shaking at 37C for 16 hr. Saturated cultures were then pooled and DNA extracted in a single midiprep reaction (QIAGEN Plus Midi Prep Kit) using one column per plate and pooling DNA from duplicates after purification. A total of 30 mg (approximately 100 ng of each vector, in triplicate), including spike-in DNA of control vectors, was digested overnight with NotI to release the targeting vector from the linear plasmid backbone. The universal control vectors included in each transfection were as described previously (Gomes et al., 2015). Briefly, four sexual stage specific genes (p25, p28, p230p and soap) had wild-type growth phenotypes and their weighted mean growth rate on a given day was defined as 1. Three additional controls were chosen for their known reduced growth phenotypes (Gomes et al., 2015). Identification numbers for all vectors included in this study are shown in Table S1 and can be used to access details of each vector design, including quality control data, through the PlasmoGEM database (http://plasmogem.sanger.ac.uk). Following restriction digests the vector pools were purified by ethanol precipitation and DNA for each triplicate pool was resuspended in 18 mL of nuclease-free water. Rat derived P. berghei schizonts were harvested after 22 hr in culture and purified on a Histodenz (Sigma) gradient. Purified parasites were pelleted at 450 g for 3 min, resuspended in 54 mL P3 Primary Cell 4D-Nucleofector solution (Lonza) and added to the DNA solution. Of this mixture exactly 26 mL were transferred into each of three separate wells of a 16X 4D-Nucleofector strip cuvette. Cells were electroporated using the FI115 program on the 4D-Nucleofector core system equipped with an X Unit (Lonza). Transfectant parasites were injected intravenously into mice and selected with 0.07 mg / mL pyrimethamine in drinking water (pH 4.5) from day one post infection (p. i.).