Overview of the magic pool strategy.

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Overview of the magic pool strategy. Overview of the magic pool strategy. (A) Basic structure of a typical transposon delivery vector (not drawn to scale). The inverted repeat (IR) for the specific transposase is indicated. We dissected the transposon delivery vector into five different parts compatible with Golden Gate assembly, and the different parts are indicated by different colors. (B) General workflow of construction and application of magic pools. In step 1, variants of the five different parts are designed, cloned into a part-holding vector, confirmed by sequencing, and archived. In step 2, the part vectors are mixed and assembled using Golden Gate assembly to produce the magic pools of transposon delivery vectors. In step 3, the magic pool vectors are characterized by DNA sequencing whereby each unique DNA barcode (random 20-nucleotide DNA barcode [N20]) is linked to a specific combination of parts. In step 4, preliminary mutant libraries of approximately 5,000 CFU are made using the magic pool, and TnSeq is performed to link the DNA barcode to the insertion site, thereby simultaneously assessing the efficacy of the vectors in the magic pool. ID, identification. In step 5, an effective vector is reassembled using the archived parts, fully barcoded with millions of random DNA barcodes, and a full RB-TnSeq transposon mutant library is constructed. oriT is the origin of transfer. AmpR is the beta-lactam resistance cassette. R6K is the conditional replication origin. Hualan Liu et al. mSystems 2018; doi:10.1128/mSystems.00143-17