Quickchange lightning

cDNA for human MTHFD2 was obtained from the Dana-Farber/Harvard Cancer Center DNA Resource Core (HsCD00040365). This was amplified via PCR to append BP sites, as well as the first 18 nt of MTHFD2 missing from the DF/HCC cDNA construct. It was introduced into pDONR221 via Gateway BP reaction (Invitrogen). Synonymous point mutations were made using Agilent QuikChange Lightning, according to the manufacturer’s protocols, to confer RNAi resistance to two shRNA hairpins (Clone ID TRCN0000036553, Clone name NM_006636.2-772s1c1, Target sequence 5′-GCAGTTGAAGAAACATACAAT-3′; and Clone ID TRCN0000036551, Clone name NM_006636.2-1067s1c1, Target sequence 5′-CGAGAAGTGCTGAAGTCTAAA-3′), and to add a stop codon, yielding the cDNA construct for MTHFD2. To generate dehydrogenase-dead MTHFD2, Agilent QuickChange Lightning was used on the previously described plasmid to mutate the Arginine in the MTHFD2 protein sequence KNVVVAGRSKN to Alanine18 (link). Mitochondrial-targeted eGFP in pDONR221 was obtained from the Mootha Laboratory. All three cDNAs for overexpression were cloned into PCWPUROCV5[ccdB] via LR reaction (Invitrogen).
MTHFD2-3xFLAG was constructed by deleting the stop codon in the cDNA encoding human MTHFD2 (Origene) and then ligated into CMV14-3xFlag (Sigma Aldrich) using standard PCR-based cloning. Correct sequences were verified using DNA sequencing.

All strains used in this study are summarized in Table S3. Bacterial clinical isolates deposited in the University of Zurich repository were screened for resistance-gene annotations to identify three isolates with a gene annotation for only aac(3)-IV, i.e., no other aminoglycoside-resistance gene that might interfere with the phenotypic interpretation of aac(3)-IV. A set of E. coli strains recombinantly expressing aac(3)-IV under defined constitutive promoter control was engineered as previously described [11 (link)]. In brief, a synthetic aac(3)-IV gene was cloned into a low-copy number plasmid with pBR322 origin of replication, an ampicillin-resistance cassette, and a T7 terminator sequence. Gene expression was controlled by an insulated constitutive promotor of defined strength. The −10 hexamers gtatct (+), taggct (++), and taatat (+++) were chosen to provide a range of promoter strengths increasing by about half-log increments, defined as relative promoter units (RPUs) 0.009, 0.030, and 0.119 in the original publication [37 (link)]. Mutant genes were constructed by PCR mutagenesis (Agilent Quick Change Lightning).

Site directed mutagenesis was performed using Quickchange lightning (Agilent) according to the manufacturer's protocol. Primers used for mutagenesis are shown in Supplementary Table S5. PCRs were Dpn1 treated and used to transform E. coli DH5α. For the LARP1 R840E/Y883A double point mutant, mutagenesis was performed sequentially post sequencing of the correct clone.

p53 mutants were created by site-directed mutagenesis (QuickChange Lightning, Agilent technologies, Santa Clara, California, USA) according to the manufacturer instructions. p53, p63 and p73 isoforms were generated by PCR (Supplementary Table S1).