Nb bsmi

The integrative vectors (Additional file 1: Table S1) were constructed by USER fusion [98 ]. The particular BioBricks (Additional file 1: Table S2) were amplified by PCR with Phusion U polymerase (ThermoFisher Scientific) under the following conditions: 98 °C for 2 min, 30 cycles of 98 °C for 10 s, 54 °C for 10 s, 72 °C for 30 s/1 kb, 72 °C for 10 min. Used templates and primers are listed in Additional file 1: Tables S1 and S3. DNA fragments were gel purified and incubated in HF buffer together with USER enzyme (New England BioLabs) for 25 min at 37 °C, followed by incubation at 25 °C for 25 min. The reactions were transformed into chemically competent E. coli cells. Empty integrative vectors were digested with FastDigest SfaAI (ThermoFisher Scientific) restriction endonuclease, nicked with Nb.BsmI (New England BioLabs) and assembled with PCR amplified genes and promoter(s) of choice. Heterologous genes were either synthetized by GeneArt or amplified from the genomic DNA of the organisms of origin (Additional file 1: Table S2). Single gRNA vectors were constructed via the whole plasmid amplification as described in [99 (link)] using the primers listed in Additional file 1: Table S3. Multiple gRNA vectors were constructed via assembly of several single gRNA expression cassettes into a USER site containing 2-µm vector as described in [100 (link)].

We used norfloxacin to inhibit wild-type DNA gyrase and topoisomerase IV (Topo IV) in vivo as described elsewhere (27 (link),28 (link)). Drug-resistant mutants allowed us to selectively inhibit DNA gyrase or Topo IV. Cells were treated with 15 or 156 μM Norfloxacin for 15–30 min just before harvest. In all cases the DNA samples analyzed were prepared from separately isolated pools of supercoiled DNAs mixed in vitro with nicked forms of knotted or catenated molecules.
To induce single-stranded breaks, DNA was digested with Nb.BsmI (New England Biolabs) for 1 h at 8 U/μg of DNA at 50ºC. Reactions were blocked with 100 μg/ml proteinase K (Roche) for 30 min at 37ºC (29 (link)).

USER cloning^{37 (link)} was used to construct plasmids, unless otherwise specified, and the EasyClone MarkerFree system with integration plasmids and gRNA plasmids compatible with CRISPR/Cas9^{38 (link)} were used throughout. USER-compatible vectors were treated with FastDigest SfaAI (Thermo Fisher Scientific) and Nb.BsmI (NEB) prior to ligation, and USER-compatible fragments were amplified with Phusion U Hot Start PCR Master Mix (Thermo Fisher Scientific) to read through uracil overhangs contained in oligos. Strains, plasmids, oligos, gene blocks, and heterologous GPCR accession IDs are listed in Supplementary Data 4–8.

The integrative plasmids (Table S7) were constructed using gene and promoter BioBricks (Table S8). Specific primers (Table S8) were used to amplify the fragments using Phusion U polymerase (ThermoFisher Scientific, Waltham, MA, USA). The native genes were amplified from S. cerevisiae CEN.PK genomic DNA, and the heterologous genes were synthetized by GeneArt (ThermoFisher Scientific, Waltham, MA, USA). The empty integrative vectors were digested with FastDigest SfaAI (ThermoFisher Scientific, Waltham, MA, USA) restriction endonuclease, nicked with Nb.BsmI (New England BioLabs, Ipswich, MA, USA), and finally assembled together with the PCR-amplified genes and promoters. To express the transporter coding gene in the oocytes, it was cloned downstream of the T7 promoter in the USER compatible Xenopus expression vector pNB1u as described previously [2 (link),3 (link)]. The empty vector was digested by PacI and nicked by Nt.BbvCI (New England BioLabs, Ipswich, MA, USA) for USER-cloning of the amplified transporter ORF. The reaction was transformed into chemically competent E. coli cells. Finally, plasmid DNAs from single colonies were sequenced and confirmed.

For the fully ssDNA construct, an 8.1 kbp dsDNA construct with digoxigenin (DIG) and biotin labeled ends with a free 3′ end was constructed as previously described (Figure 1A) (Naufer et al., 2017 (link)). The dsDNA vector pBacgus11 (gift from Borja Ibarra) was linearized using high fidelity restriction enzymes BamHI and SacI (New England Biolabs). A dsDNA handle with digoxigenin (DIG) labeled bases with a complementary end to the BamHI sequence was PCR amplified (Ibarra et al., 2009 (link)). The DIG handle and a biotinylated oligonucleotide with a 3′ end complementary to the SacI sequence (Integrated DNA Technologies) were ligated to the linearized pBacgus11 using T4 ligase (NEB). For the dsDNA/ssDNA hybrid construct, a 6.5 kbp fragment contained in a plasmid (gift from Tom Perkins) containing a designed sequence (Okoniewski et al., 2017 (link)) was PCR amplified using KOD Hot Start DNA Polymerase (Novagen). One end of product contains a biotinylated primer, and the other end was cut and ligated to same DIG handle described above. The labeled construct was then nicked using restriction enzymes Nt.BspQI and Nb.BsmI (New England Biolabs).

Plasmid construction was performed based on the EasyCloneYALI toolbox described by Holkenbrink et al. (2018 ). Integrative and gRNA vectors were constructed and used to obtain chromosomal integration of expression cassettes in defined genomic loci of Y. lipolytica. Lists of primers, synthetic genes, biobricks, and plasmids used in this study are provided in Tables S1–S4 (Supporting Information), respectively. Biobricks were amplified by using Phusion U polymerase under the following conditions: 98°C for 5 min, 30 cycles of (98°C for 20 s, 54°C for 30 s, and 72°C for 30 s/kb), 72°C for 7 min. PCR products were purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). Assembly of plasmids was performed by USER^® cloning. Parent vectors were treated with FastDigest SfaAI (Thermo Fisher Scientific) restriction enzyme and Nb.BsmI (New England BioLabs) nicking endonuclease. Opened vector and compatible biobricks/primers were transformed into Escherichia coli strain DH5α. The correct assembly of plasmids was confirmed by colony PCR and Sanger sequencing.