A SARS-CoV-2 plasmid standard (2019-nCoV_N_Positive Control, 4.12 kbp, catalog number 10006625, Integrated DNA Technologies, Coralville, Iowa, USA), including both the N1 and N2 genes, was linearized by enzymatic digestion with ScaI-HF (New England Biolabs, catalog number R3122S). Briefly, 10 µl of the plasmid was incubated with 5 U of ScaI-HF in a 50 µl reaction at 37°C for 60 min. The enzyme was inactivated at 80°C for 20 min. The BCoV standard was synthesized as a DNA Ultramer (Table S3, Supporting Information ; Integrated DNA Technologies). Standards for SARS-CoV-2 and BCoV were assayed in triplicate to generate a standard curve for the three targets (Figure S3, Supporting Information ).
Scai hf
Manufactured by New England Biolabs
Sourced in United States
ScaI-HF is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 'AGTACT' with high fidelity. It is a recombinant enzyme derived from the Streptomyces scalarisporus bacterium.
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Lab products found in correlation
Qiaquick pcr purification kit, by Qiagen (3 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Kod hot start master mix, by Merck Group (2 mentions)
Sphi hf, by New England Biolabs (2 mentions)
Hybond, by Cytiva (2 mentions)
Dna clean and concentrate kit, by Zymo Research (1 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions)
Trizol extraction kit, by Thermo Fisher Scientific (1 mentions)
Bsrgi, by New England Biolabs (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Dna loading dye, by New England Biolabs (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Dig luminescent detection kit, by Roche (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Draiii hf, by New England Biolabs (1 mentions)
α 32p dctp, by PerkinElmer (1 mentions)
16 protocols using scai hf
1
SARS-CoV-2 and BCoV Plasmid Standards
2
SARS-CoV-2 and BCoV Plasmid Standards
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A SARS-CoV-2 plasmid standard (2019-nCoV_N_Positive Control, 4.12 kbp, catalog number 10006625, Integrated DNA Technologies, Coralville, Iowa, USA), including both the N1 and N2 genes, was linearized by enzymatic digestion with ScaI-HF (New England Biolabs, catalog number R3122S). Briefly, 10 µl of the plasmid was incubated with 5 U of ScaI-HF in a 50 µl reaction at 37°C for 60 min. The enzyme was inactivated at 80°C for 20 min. The BCoV standard was synthesized as a DNA Ultramer (Table S3, Supporting Information ; Integrated DNA Technologies). Standards for SARS-CoV-2 and BCoV were assayed in triplicate to generate a standard curve for the three targets (Figure S3, Supporting Information ).
3
Bacillus subtilis Riboswitch Constructs
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All B. subtilis reporter strains were derived from KK642 (Em his nprE18 aprE3 eglSΔ102 bglT/bglSΔEV lacA::PxylA-comK loxP-Pveg-btuFCDR queG::loxP) which was derived from strain 1A976 of Zhang et al. (16 (link), 30 (link)). All riboswitch mutant constructs were ordered as eBlocks from IDT (Benchling links in Table S1). Each was designed to contain the full-length riboswitch with homology to pKK374 at the NheI (NEB) cut site (16 (link)). Linearized pKK374 and the eBlocks were assembled via Gibson assembly. Plasmids were then transformed into XL1-Blue competent cells (UC Berkeley Macrolab) and plated on LB with 100 µg/mL ampicillin. Plasmids from three or four colonies were purified and Sanger sequenced at the Barker DNA Sequencing facility. Plasmids with the correct sequence were linearized with ScaI-HF (NEB) and transformed into the B. subtilis fluorescent reporter strain KK642 where they were integrated into the chromosome at the amyE locus and plated on LB with 100 µg/mL spectinomycin. Successful integration was confirmed by PCR.
4
Cloning and sequencing of DNA fragments
5
Recombination of MhTHI4 into Plasmid p1
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Ten micrograms of plasmid GR-306MP_MhTHI4 were digested overnight using 1.5 µL of ScaI-HF (New England Biolabs, catalog no. R3122) in a total volume of 50 µL, then heat-inactivated for 20 min at 65°C. A 2-µL aliquot was run on a 1% w/v agarose gel to check for complete digestion, and the remaining 48 µL was used to transform strain GA-Y319 as above. Transformants were selected on SC-Leu for 5 d at 30°C. To confirm recombination of MhTHI4 into p1, single colonies were inoculated into 5 mL SC-Leu and grown to saturation for ∼2 d at 30°C and 220 rpm. Total DNA was isolated as above and 1 µg was run on a 0.8% w/v agarose gel to verify the presence of p1_wt, p1_rec, and p2_wt. The presence of the 10B2 promoter, MhTHI4, and the poly(A) tail was checked by PCR using total DNA as template and primers p1_F and Ribozyme_R (Supplemental Table S2 ) and sequencing of the amplicons.
6
Genotyping F2 Progeny via Restriction Digest
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Red fluorescent F1 progeny (n = 24 total), from successfully injected P0s, were singled to 35 mM NGM dishes and allowed to lay F2 eggs for 2 d. After egg-laying, individual F1s were placed in 7 μl of PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl pH = 8.3, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween-20) containing 1 mg/ml proteinase K in PCR strip tubes. After 1 hr at −80°, the PCR tubes were lysed for 1 hr at 60°, followed by 15 min at 95° to inactivate the proteinase K. An aliquot (4 μl) of the genomic DNA containing single worm lysate was used to PCR amplify an 820 bp fragment surrounding the targeted genomic edit using Q5 Hot Start High-Fidelity 2X Master Mix (NEB). The primer sequences for lgc-35 were: F1: 5′-TCGTCATTACGTCCTGGGTTTC-3′ and R1: 5′-CCATTGGTTCAAGACGGGTAAG-3′. PCR products were purified and eluted in 10 μl nuclease-free water using a DNA Clean and Concentrate kit (Zymo Research). The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.
7
Chromatin Reconstitution and Analysis
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Nucleosomes and chromatin arrays were reconstituted on a scale from 10 to 100 pmol (per mononucleosome) in 25–30 μl. 601 NPS DNA (1 or 12 repeats) was reconstituted in TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5) and 2 M NaCl, followed by the addition of 1.1 equivalents of the respective histone octamers. For chromatin arrays, 0.5 equivalents of MMTV buffer DNA was included31 (link). The reactions were dialysed gradually from TEK2000 buffer (TE buffer including 2000, mM KCl) to TEK10 buffer over 16 h, using a two-channel peristaltic pump. After dialysis, chromatin concentrations were determined by UV quantification. 0.5–2 pmol of chromatin arrays were digested with ScaI-HF (NEB) in 10 μl for 6 h. Quality of the reconstitution was assessed by native PAGE (Supplementary Fig. 3b,c ). Chromatin and nucleosome reconstitutions were stored on ice for maximum 1 week after preparation.
8
Whole-cell RNA Extraction and Analysis
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We collected whole-cell RNA from conjugating cells at six developmental time points (0, 6, 12, 18, 36, and 60 h post-mixing) using a TRIzol extraction kit (Invitrogen). We DNase-treated (Turbo DNAse) 10 µg RNA, precipitated it in phenol–chloroform, and resuspended it in 24 µL nuclease-free water (Agencourt Bioscience Corp., Beverly, MA). Reverse transcription reactions were performed with Superscript III enzyme on 3 µg RNA, using a telomeric primer (5′-CCCAAAACCCCAAAACC-3′). In addition, control reactions at each time point without reverse transcriptase showed no qualitative product (Fig. 9 ), but were sequenced as negative controls. For PCR we used eight replicates per time point to reduce jackpot effects. Fragments were amplified using FastStart enzyme with 0.5 µM of the same telomeric primer used for reverse transcription through 40 cycles of touchdown PCR (70°–55° for 30 cycles, then 10 cycles at 55°). Replicates were pooled and digested with BsrGI and ScaI-HF (NEB) to eliminate contaminating hexamer repeats observed in previous experiments (data not shown).
9
Characterization of AdeRS system in A. baumannii
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The adeRS backbone of the reference strain ACICU (30 (link)) was used since it represents the wild type of the globally spread A. baumannii international clone 2 (IC2) and was therefore more appropriate for characterization of amino acid substitutions identified in recent clinical isolates than the uncommon adeRS configuration of ATCC 17978 (23 (link)). Genomic DNA was extracted from a heat-induced crude cell lysate. Amplification of the target DNA was performed using Q5 high-fidelity DNA polymerase (New England BioLabs, Frankfurt, Germany). PCR settings were adjusted according to the PCR product size and the nucleotide sequence of the primer pair O47-O48 (Table S1 in the supplemental material). PCR products were purified using the QIAquick PCR purification kit (Qiagen). The shuttle vector pJN17/04 (21 (link)) was linearized by the restriction enzyme ScaI-HF (New England BioLabs). The In-Fusion HD cloning kit (TaKaRa Clontech, Saint-Germain-en-Laye, France) was used for directional cloning of adeRS into the shuttle vector. Cloned plasmids were transferred into chemically competent Escherichia coli HST08 cells via heat shock according to the manufacturer’s instructions.
10
Programmable DNA cleavage by KmAgo
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In two half-reactions, 8 pmol of KmAgo was loaded with either 10 pmol of forward or reverse DNA guide in reaction buffer containing 5 mM HEPES–NaOH pH 7.5, 50 mM NaCl, 0.5 mM MnCl2, 2.5% glycerol. The half-reactions were incubated for 30 min at 55°C. Next, both half-reactions were mixed and 200 ng target plasmid was added, after which the mixture was incubated for 2 h of 37°C. After the incubation, NdeI (NEB) or ScaI-HF (NEB) and Cutsmart buffer (NEB) were added and incubation for 2 h at 37°C. A 6× DNA loading dye (NEB) was added to the plasmid sample prior to resolving it on a 1.0% agarose gel stained with ethidium bromide. In the double-stranded assays in which linear plasmid was targeted, 200 ng pUC19, linearized by NdeI, was added to both half-reactions and incubated for 2 h at 37°C. The reaction products were resolved on a 1.0% agarose gel.
For KmAgo–gDNA duplex formation and HIV-1 ΔDIS 5′UTR cleavage, KmAgo (800 nM) was pre-mixed with gDNA (400 nM) for 10 min at 37°C in a 9 μl reaction followed by addition 1 μl of HIV-1 ΔDIS 5′UTR substrate (final concentration: 250 nM) and incubation at 37°C for 15 min. Reactions were quenched with 2× RNA loading dye and heating it for 5 min at 95°C. The cleavage products were resolved by 20% denaturing PAGE, stained with SYBR Gold, and visualized with Gel Doc™ XR+ (Bio-Rad).
For KmAgo–gDNA duplex formation and HIV-1 ΔDIS 5′UTR cleavage, KmAgo (800 nM) was pre-mixed with gDNA (400 nM) for 10 min at 37°C in a 9 μl reaction followed by addition 1 μl of HIV-1 ΔDIS 5′UTR substrate (final concentration: 250 nM) and incubation at 37°C for 15 min. Reactions were quenched with 2× RNA loading dye and heating it for 5 min at 95°C. The cleavage products were resolved by 20% denaturing PAGE, stained with SYBR Gold, and visualized with Gel Doc™ XR+ (Bio-Rad).
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