Hifi assembly master mix

Plasmids expressing GFP-tagged, Fc-tagged, or FLAG-tagged SPIN1, WT, or mutant SPIN4 were constructed by PCR amplification of gene fragment using Q5 High-Fidelity DNA Polymerase (New England Biolabs), followed by Gibson assembly using HiFi assembly master mix (New England Biolabs). The plasmids used for the backbone included pEGFPN1 (Clontech), pSecTag (Invitrogen), and pcDNA3.1 (Invitrogen). Plasmid expressing H2B-GFP was a gift from Geoff Wahl (The Salk Institute for Biological Studies, La Jolla, California, USA; Addgene, plasmid no. 11680) (34 (link)). TOPFLASH and FOPFLASH plasmids were a gift from Randall Moon (University of Washington, Seattle, Washington, USA; Addgene, plasmid nos. 12456 and 12457) (22 (link)). pcDNA3-HA-TCF1 was a gift from Kai Ge (NIDDK; Addgene, plasmid no. 40620) (35 (link)).

Restriction enzymes, T4 ligase, Dulbecco’s modified Eagle medium (DMEM), foetal bovine serum, Turbofect and penicillin/streptomycin were purchased from Thermo Fischer Scientific Inc. DNA oligonucleotides and the GenElute HP Plasmid Miniprep and Midiprep kit used for plasmid purifications were acquired from Sigma-Aldrich. Q5 High-Fidelity DNA Polymerase, NEB5-alpha competent cells and HiFi Assembly Master Mix were purchased from New England Biolabs Inc.

The transposable element (TE) insertion was amplified from s¹ genomic DNA using LongAmp polymerase (New England Biolabs) with tailed primers gibpg7-Yippee-5′-Region-F1 gcggccgcgggaattcgattCCGGGCAGCCACGCAAGGATTGCAT and gibpg6-Yippee-5′region-R2 ccgcgaattcactagtgattGGTCAGGTGTCCGGTGTCAGGG. The ∼8 kbp PCR band was gel purified and assembled into pGem-T-Easy using HiFi Assembly Master Mix (New England Biolabs). Three clones were fully sequenced using Oxford Nanopore technology by Plasmidsaurus (Eugene, OR), then aligned to generate a consensus sequence.

Strains and plasmids are listed in Table S1, and primers are listed in Table S2 in the supplemental material. E. coli strain DH10B was used as a host strain for cloning, and confirmed plasmids were subsequently electroporated into S. Typhimurium. The def gene was cloned into the EcoRI and KpnI sites of pTrc99A to generate pAS18 (pdef) and into the BamHI and EcoRI sites of pGEX-2T to generate pAS19. A C-terminal HA tag was introduced in the coding region of def using oligonucleotides ASP62 and ASP35, and the amplified product was cloned into pTrc99A to generate pAS33. Site-specific mutagenesis of PDF-Cys90 was performed using HiFi assembly master mix (New England BioLabs) to generate pAS37 and pAS40. All plasmids were confirmed by DNA sequencing (Genewiz).

To generate FlipGFP-based reporters, the TEV FlipGFP plasmid PCDNA3-FlipGFP(TEV cleavage seq) T2A mCherry (Addgene, #124429, a gift from Xiaokun Shu [9 (link)]) was used as a template for pairs of PCR reactions including primers designed to generate overlapping products replacing the TEV cleavage site with the indicated cleavage sequence (S1 Table). For example, to replace the TEV cleavage site with the PLP2 cleavage sequence, products generated by PCR reactions including ‘Near AfeI’/‘PLP2 Rv’ and ‘PLP2 Fw’/‘Near AflII’ primer pairs were used. The same plasmid was then digested with AfeI and AflII and assembled with the gel purified PCR products using the HiFi Assembly Master Mix (NEB, E5520).
For MPro, we selected a wildtype self-cleavage sequence (SAVLQ/SGF, herein termed WT3c) present between nsp4 and nsp5, and an “optimal” cleavage sequence (TVRLQ/SGF, herein termed Opt3c) found by substrate profiling of SARS-CoV MPro [30 (link)]. For PLPro, we selected all three cognate cleavage sequences present in the pp1a polyprotein (ELNGG/AYT, herein termed PLP1; TLKGG/APT, herein termed PLP2; and ALKGG/KIV, herein termed PLP3).
To generate a non-cleavable Opt3c-FlipGFP (MPro) reporter, the critical glutamine residue in the cleavage site was changed to isoleucine (TVRLI/SGF). For the PLP2-FlipGFP (PLPro) reporter, the critical LKGG sequence was scrambled to GLGK (TGLGK/APT).

Mutants were created by allelic exchange in strain TIGR4 using the EF3030-TIGR4 orthologous genes. Mutagenic PCR constructs were generated by amplifying the upstream and downstream DNA fragments flanking the gene(s) of interest followed by fusion of these fragments with the Janus Cassette using a HiFi assembly master mix (NEB). Transformation of TIGR4 with the mutagenic construct (100ng/ml) was performed by natural transformation which was induced using competence-stimulating peptide variant 2 (CSP-2) as previously described (36 (link)). Blood agar plates supplemented with kanamycin (300 mg/L) were used for selection.