Extended Data Fig. 1: Steps in REXER-mediated integration of ~100 kb of synthetic DNA into the E. coli genome using homology region (HR)-specific spacers.
From: Continuous synthesis of E. coli genome sections and Mb-scale human DNA assembly
REXER allows integration of more than 100 kb of synthetic DNA (pink) into the genome, through replacement of the corresponding genomic DNA. A bacterial artificial chromosome (BAC) containing the synthetic DNA of interest is electroporated into competent cells with a suitably marked genome, the cells also contain a helper plasmid encoding the Cas9 protein and the lambda red recombination components. Selection for the helper plasmid (+5) and the BAC (+2) is applied. A clonal cell is then expanded and induced with arabinose to express the helper plasmid genes and made electrocompetent again. HR-specific spacer arrays (either plasmid-based as shown, or as linear DNA) are then electroporated into the cell; this leads to CRISPR/Cas9 mediated in vivo excision of the synthetic DNA, flanked by a double selection cassette (+2/−2) and HRs to the genome, from the BAC. The lambda red recombination machinery then uses the HRs to direct the integration of the excised DNA into the genome. Triangles denote the Cas9 cleavage sites at the HRs (grey boxes) flanking the synthetic DNA. Selection on tetracycline (maintenance of +5), ampicillin (maintenance of +6), chloramphenicol (maintenance of +2), and streptomycin (loss of −1) ensures only cells where the recombination took place over the whole section survive. The selectable markers are +1, blue, kanR (selected for with kanamycin); −1, yellow, rpsL (selected against with streptomycin); +2, green, cat (selected for with chloramphenicol); −2, pink, sacB (selected against with sucrose); +5, dark blue tetR (selected for with tetracycline); +6, red ampR (selected for with ampicillin).
