Extended Data Fig. 5: Identification of D7-ZF.

a Overview of the heterodimer positive and counter-selection and the clone selection procedure. Plasmid-based activity assays are shown for the D7 recombinase containing the ZFL and ZFR libraries on the loxF8 and loxF8-flank target sites. Activities of the initial and the final library after 8 cycles of counter-selection on loxF8 and 3 cycles of positive selection on loxF8-flank are shown. 100 ug/ml L-arabinose was used for the initial library on loxF8-flank, 200 ug/ml L-arabinose for the initial library on loxF8 and for the final library on loxF8 and loxF8-flank. After the selection, single clones were picked and their activity was analysed by PCR. b Schematic representation of the PCR-based recombination test used for single clone analyses (adapted from Lansing et al.⁸). Primers are indicated as coloured arrows. Three-primer PCR generates a bigger fragment (491 bp) from the non-recombined pEVO plasmid, or a smaller fragment (400 bp) from the recombined plasmid, if a mix of both plasmids used for the PCR both bands are detected. c 95 heterodimer D7-ZF clones were expressed on loxF8 target site in E.coli and analysed by the PCR-based recombination test (n = 1). Clones that did not recombine the loxF8 target site and were selected for further analysis and are marked with black triangles. d Plasmid-based activity assay for the candidate D7-ZF clones on the loxF8 and loxF8-flank. 200 ug/ml L-arabinose was used for the test on loxF8 and 10 ug/ml L-arabinose on loxF8-flank. Recombination efficiencies were calculated from ratios of recombined and non-recombined band intensities. Activity of the most efficient clone (D7-ZF, G10) that was selected for future experiments is highlighted by a green box. The assay was performed three times (n = 3 biologically independent replicates). For (a-d), the upper band represents the unrecombined plasmid (line with two triangles), the lower band represents the recombined plasmid (line with one triangle). M = Marker. e Amino acid sequence of the selected monomers of the D7-ZF (G10, one-letter code). The sequence of the ZFL is highlighted in orange, the sequence of the ZFR is highlighted in blue, the sequence of the linkers is highlighted in grey. Asterisks denote stop codons.