Fig. 4: Recombination and cross-reactivity of LoxPsym variants in E. coli and Z. mays.

a Experimental design for determining LoxPsym variant cross-reactivity in E. coli. A donor (full line) and acceptor (dashed line) plasmid were co-transformed, each carrying one LoxPsym variant (differently colored diamonds) and in vivo recombination was verified using PCR. b Acceptor plasmid encodes the Cre gene, controlled by rhamnose inducible rhaB promoter and rrnB terminator. Induction of Cre expression (4 h) results in recombination only when LoxPsym variants are cross-reactive, in which case an amplicon will be generated by PCR (blue arrows). Note that the recombination reaction does not have a final state because recombined plasmids can recombine back to separate plasmids. c Recombination efficiencies between LoxPsym-NNN variants in E. coli, calculated from densitometric analysis of the junction PCR. Data represent band intensities of PCRs performed in technical duplicate, using a mixture of templates derived from three biological replicates. d Experimental design for determining LoxPsym variant cross-reactivity in Z. mays. The combinatorial library that included all 256 pairwise combinations of LoxPsym variants was transfected to Z. mays protoplasts together with a plasmid for constitutive Cre expression. Occurrence of recombination was verified using NGS. e Each plasmid of the combinatorial library encoded two LoxPsym variants (differently colored diamonds), separated by a 104 bp linker (gray) that contains recognition sites (RE1 and RE2) for restriction enzymes NcoI-HF and PvuI-HF (dashed lines). Barcodes were incorporated up- and downstream of LoxPsym variants, with each barcode uniquely linked to one LoxPsym variant. The library contained all 16 × 16 (=256) combinations between LoxPsym variants. Blue arrows indicate primer annealing sites for the PCR that was carried out after recombination induction. PCR amplicons were analyzed by NGS. f Recombination efficiencies between LoxPsym-NNN variants in Z. mays, calculated from the abundance of sequenced reads. Note that all efficiencies were normalized to the most active recombination site, LoxPsym-GGC, for which the efficiency was arbitrarily set to 100%. Data represent the average of three technical repeats for two biological replicates, shown separately by diagonally split cells. Source data are provided as a Source Data file.