Gibson master mix

DNA was extracted from cells using either a Qiagen (Hilden, Germany) or a Zymo Research (Irvine, CA) Miniprep kit according to the manufacturer's instructions. Polymerase chain reaction (PCR) was used to amplify genes or DNA of interest using Q5 DNA Polymerase (NEB). Primers were ordered from Integrated DNA Technologies (IDT, Coralville, IA). NEB restriction enzymes such as BamHI and HindIII were used to generate restriction digests of desired PCR products or plasmids. Agarose gel electrophoresis was used to separate DNA fragments based on size and the gel bands (as visualized with SYBR Safe, Invitrogen) as well as DNA sequencing by Genewiz was used to verify the constructs. Digested fragments were ligated using either NEB Quick Ligase or NEB T4 Ligase. Gibson Assembly was performed with NEB's Gibson Master Mix according to the manufacturer's instructions. Electro- or chemically competent cells (either from NEB, Invitrogen (Carlsbad, CA), electrocompetent, or made with Zymo Research's Z- Competent E. coli Transformation Kit) were used for transformation.

All PCRs were conducted using Phusion polymerase (NEB) using primers listed in Dataset S2. Constructs were assembled using Gibson master mix (NEB). Point mutations were created by the Phusion mutagenesis protocol. AC9^AID tagging constructs were generated using a PCR-amplified homology-directed repair template (P1-2) and a CRISPR-Cas9 pU6-Universal plasmid with a protospacer against the 3′ untranslated region of the gene of the interest (P3-4). AC9-BirA*, ERK7, and AC10 C-terminal tagging constructs were generated using P1-4, P5-8, and P9-12, respectively. To generate the wild-type complementation construct, the complete AC9-coding sequence was PCR-amplified from complementary DNA and cloned into a UPRT-locus knockout vector driven by the ISC6 promoter. Both the 3×Glu and the phosphorylation mutants were constructed using synthetic genes (Quintara Biosciences) and cloned into the pISC6-UPRT vector (Dataset S2).

The oligonucleotides used in this study are listed in Table 3. DNA ligations, restriction endonuclease digestions, and agarose gel electrophoresis were performed using standard molecular biology techniques (81 ), with Gibson assembly undertaken according to published protocols (82 (link)). All restriction enzymes, T4 DNA ligase, and Gibson master mix were used as recommended by the manufacturer (New England Biolabs). E. coli PIR2 and DH5α cells were transformed using heat shock-based transformation. PCR amplifications were carried out using either Phusion DNA (Thermo Fisher Scientific) or Pfu Ultra II (Agilent) polymerases were used according to the manufacturer’s recommendations with the addition of 2.5% dimethyl sulfoxide (DMSO) for the amplification of B. cenocepacia DNA due to its high GC content. DNA isolation, PCR recoveries, and restriction digest purifications were performed using the genomic DNA cleanup kit (Zmyo Research, CA) or Wizard SV gel and PCR cleanup system (Promega). Colony and screening PCRs were performed using GoTaq Taq polymerase (Qiagen) supplemented with 10% DMSO when screening B. cenocepacia. All constructs in Table 2 were confirmed by Sanger sequencing undertaken at the Australian Genome Research Facility (Melbourne, Australia).

All PCRs were conducted using Q5 DNA polymerase (New England BioLabs) and the primers listed in Table S1 in the supplemental material. Constructs were assembled using Gibson master mix (New England BioLabs).