Monarch dna kits

Oligoduplex primers (Supplementary Table S1) were synthesized by IDT. Molecular biology reagents including Q5 Hot Start High-Fidelity DNA polymerase, NEBuilder HiFi DNA assembly master mix, restriction enzymes, DNA size standards, lambda DNA, and competent cells were from New England Biolabs (NEB). Plasmid purification and nucleic acid purification clean ups were performed using Monarch DNA kits (NEB). Plasmid DNA constructs were confirmed by sequencing on an ABI 3130xl capillary machine (Applied Biosystems).

The chromosomal DNA of P. aeruginosa NCTC 11442 was extracted using the GenElute Bacterial Genomic DNA kit (Merck). Plasmid backbones and inserts for cloning were amplified from the purified chromosomal DNA using Q5 High-Fidelity DNA Polymerase (NEB). PCR products and plasmids were purified with Monarch DNA kits (NEB). Overlapping primers for amplification were designed using the NEBuilder assembly tool (https://nebuilderv1.neb.com/) (S12 Table). Plasmids and inserts were assembled using NEBuilder HiFi DNA Assembly (NEB), followed by incubation at 50°C for 20 min. Point mutations or small deletions in pGM34 and pGM71 (S11 and S12 Tables) were performed using the KLD enzyme mix (NEB)

PCR amplicons and plasmids were purified using Monarch DNA kits (NEB). PCR, restriction digests, ligations, transformations and agarose gel electrophoresis were performed using standard molecular biology techniques. Constructed plasmids were confirmed via sequencing with an Abi 3370 DNA sequencer. The pSAT1-LIC-brxR⁺ expression construct adds a cleavable N-terminal His₆-SUMO tag. Primers TRB878 and TRB879 were used to amplify brxR from pEFER (gene pEFER_0020) for insertion into pSAT1-LIC (38 (link)) to produce pTRB446 via Ligation Independent Cloning (LIC) (Supplementary Table S1). Primers TRB876 and TRB877 were used to amplify brxR from pEFER which was inserted into pBAD30 (39 (link)) to produce pBAD30-his₆-brxR (Supplementary Table S1). Primers TRB1987 and TRB1988 were used to perform QuikChange (Invitrogen) mutagenesis to produce pBAD30-his₆-brxR-R17A (Supplementary Table S1).