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. 2021 Feb 2;33(4):1161–1181. doi: 10.1093/plcell/koab024

Figure 2.

Figure 2

High-throughput recombineering pipeline for generating Venus-tagged fusion proteins with the native promoter regions intact. A, On Day 1, BAC clones containing target genes are made recombineering competent by transformation with the pRed plasmid, which encodes the viral recombinogenic Redαβγ genes and recA under the control of an arabinose inducible promoter. Transformation efficiency shown on the right-hand side relates to BAC clones that yielded colonies after selection with tetracycline and chloramphenicol. Cat: the chloramphenicol resistance gene in the backbone of every BAC clone in the BAC library. B, On or before Day 2, the recombineering cassette is amplified from pLM099 using primers that contain 50-bp homology arms complementary to regions flanking the target gene (shown in orange): one >2,000-bp upstream of the annotated ATG and one at the 3′-end of the coding sequence. On Day 2, BAC-containing cells are electrotransformed with the recombineering cassette after induction with l-arabinose. Recombination between the BAC and the cassette results in a plasmid product containing the target gene in frame with CrVenus-3xFLAG and under the control of its native promoter. Efficiency shown at this stage relates to PCRs that yielded efficient amplification of the recombineering cassette. C, On Day 3, colonies containing plasmid products are isolated. Efficiency at this stage relates to the number of transformations that yielded colonies after selection with kanamycin. D, On Day 4, plasmid products are extracted from cells, screened by enzymatic digestion and confirmed by sequencing. Efficiency shown at this stage relates to correct digest patterns with single and double cutting restriction enzymes. MW, molecular weight marker. E, Overall efficiency split into number of colonies screened via restriction digest. For 74% of target regions, the correct digest pattern was observed from plasmids isolated from the first, second or third colony picked per target. For 3% of targets, analyzing >3 colonies yielded the correct product.