Oligonucleotides

1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), bezafibrate, and fenofibrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium phenobarbital was purchased from FUJIFILM Wako Pure Chemicals (Osaka, Japan). Oligonucleotides were synthesized by Macrogen (Seoul, Korea). All other reagents were obtained from FUJIFILM Wako Pure Chemical or Sigma-Aldrich unless otherwise indicated.

Bezafibrate, rifampicin, rifaximin, simvastatin, and SR12813 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligonucleotides were commercially synthesized by Macrogen (Seoul, Korea). All other reagents were obtained from FUJIFILM Wako Pure Chemical (Osaka, Japan) or Sigma-Aldrich, unless otherwise indicated.

TNT T7 Quick for PCR DNA was from Promega (Madison, WI, USA). Dog pancreas ER rough microsomes were from tRNA Probes (College Station, TX, USA). EasyTag EXPRESS³⁵S Protein Labeling Mix, [³⁵S]-L-methionine and [³⁵S]-L-cysteine, for in vitro labelling was purchased from Perkin Elmer (Waltham, MA, USA). Restriction enzymes were from New England Biolabs (Massachusetts, USA) and endoglycosidase H was from Roche Molecular Biochemicals (Basel, Switzerland). PCR and plasmid purification kits were from Thermo Fisher Scientific (Ulm, Germany). All oligonucleotides were purchased from Macrogen (Seoul, South Korea).

We introduced point mutations into DdCBE expression plasmids through site-directed mutagenesis. In brief, we amplified plasmids with mutagenesis primers before mutagenesis with the Q5 Site-Directed Mutagenesis Kit (NEB), and the results were confirmed with Sanger sequencing. For assembly of plasmids encoding interface mutant DdCBEs, the mini-prepped mutant expression plasmid was added to a solution containing module vectors (each encoding a TALE array^{22 (link)}), BsaI-HFv2 (10 U), T4 DNA ligase (200 U) and buffer in a single tube. For the TALE-free construct, instead of module vectors we used oligonucleotides (Macrogen) (forward, 5′-CTGAGTGGTAGTGGTAGTGGTTCTGG-3′; reverse, 5′-ACCGCCAGAACCACTACCACTACCAC-3′) that encode a flexible linker N-(SG)₃-C. amino acid sequences of the TALE arrays for each target are indicated in Supplementary Fig. 16. The restriction and ligation reactions were performed in a thermocycler, with 20 cycles at 37 °C and 16 °C for 5 min each, followed by final incubations at 50 °C for 15 min and 80 °C for 5 min. Ligated plasmids were chemically transformed into Escherichia coli DH5ɑ; plasmids from the transformants were subjected to Sanger sequencing to analyze the identity of the constructs. Final plasmids were midi-prepped (Qiagen) for cell transfection.

E. coli DH10-beta (NEB, #C3019H) was used for cloning and amplification of plasmids. The plasmids were constructed by T4 DNA ligase (Thermo Fisher Scientific, Waltham, MA, USA) after digestion with restriction enzymes (NEB, Ipswich, MA, USA) or using a Gibson assembly kit (NEB, USA). KOD Plus Neo DNA polymerase (Toyobo, Japan) was used for polymerase chain reaction (PCR) amplification. Oligonucleotides were chemically synthesized (Macrogen, Seoul, Korea). The attenuated strain of S. Typhimurium SHJ2037 (relA::cat, spoT::kan), renamed SL. ΔppGpp in this study [34 (link)], was transformed with plasmids by 2.5 kV electroporation (Bio-Rad, Des Planies, IL, USA). These bacteria were grown in Luria-Bertani (LB) broth or on LB agar plates supplemented with the appropriate antibiotics at 37 °C. Transformed bacterial cells were stored at −80 °C in 25% glycerol before in vitro and in vivo assessments of the NO-sensing gene switch system.

Unless otherwise stated, all products were purchased from Sigma Aldrich (Overijse, Belgium), product concentration in % are w/v, PCR products were purified by the innuPrep PCRpure Kit (Analytik Jena AG, Jena, Germany) and plasmid extraction was performed with the Qiagen Spin Miniprep kit (Qiagen, Antwerp, Belgium). Oligonucleotides were obtained from Integrated DNA Technologies (IDT, Leuven, Belgium) and sequencing was performed by EZ-Seq (Macrogen, Amsterdam, The Netherlands). In case linear DNA fragments were needed for further cloning, they were amplified using the high-fidelity PrimeSTAR HS DNA polymerase (Takara, Westburg, The Netherlands, R040A), according to the manufacturers’ instructions. Colony PCR to confirm plasmid assembly or transformations were done using Taq DNA polymerase (NEB, M0273X) according to the manufacturers’ instructions.

For the 18S rDNA gene, primer candidates for PCR preamplification of the target sequences prior to the CRISPR-Cas reaction were designed using the Primer3Plus v.2.4.2 server with the default settings and an average Tm of 60°C (Untergasser et al., 2012 (link)).³ For kDNA minicircles, primer candidates were manually searched (18–22 nt long, average Tm of 60°C) within conserved regions identified previously.
Primer candidates were aligned against the human genome using NCBI BLAST and discarded if they had more than 80% sequence identity and coverage with any human sequence. Self- and hetero-dimer formation were tested using the IDT OligoAnalyzer Tool.⁴ Primers with ΔG < −7 kcal/mol in any parameter were discarded. For the RNase P gene, primers reported previously (Curtis et al., 2018 (link); Alcántara et al., 2021a (link)) were selected. Oligonucleotides were ordered from Macrogen Inc. (Seoul, South Korea). All primer and crRNA template sequences used in this study are listed in Supplementary Table S1.