The core enzyme of cyRNAP was overexpressed in E. coli T7Express cells (New England Biolabs) cells transformed with a pET28a expression vector containing the α, β, β’1, β’2 and ω encoding genes (β and β’2 contain a Strep-tag and His-tag, respectively) (32 (link)). The cells were grown in LB media supplemented with kanamycin (50 μg/ml) at 37 °C until the OD600 was ~0.6. Afterward, the cells were induced with IPTG (1 mM) and grown overnight at 22 °C. The biomass was harvested and suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10% glycerol, 20 mM imidazole, and 1 mM β-mercaptoethanol and protease inhibitors from Roche according to the manufacturer’s instructions). The cells were sonicated, lysate centrifuged at 18 k × g, after which the supernatant was collected. The protein was purified at 4 °C sequentially through a HisTrap (5 mL) column and a Strep-Tactin XT (1 mL) column (both from Cytiva). The latter column was washed with 3 column volumes (CVs) of Buffer W (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA). The bound protein was eluted by applying 1 CV of Buffer E (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 2.5 mM desthiobiotin). The purified cyRNAP (20 μM) was assessed using SDS PAGE, dialyzed against Storage Buffer (40 mM Tris–HCl pH 8.0, 200 mM KCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol), and stored at −80 °C.
T7 express cells
Manufactured by New England Biolabs
T7 Express cells are a strain of Escherichia coli (E. coli) bacteria that have been genetically modified to facilitate the expression of proteins under the control of the T7 promoter. These cells contain the T7 RNA polymerase gene, which allows for efficient transcription of target genes cloned into T7 expression vectors.
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Lab products found in correlation
Nebuilder hifi dna assembly master mix, by New England Biolabs (1 mentions)
E coli bl21 de3, by New England Biolabs (1 mentions)
Ni nta superflow resin, by Qiagen (1 mentions)
Pgex 2tk, by Merck Group (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
Histrap hp, by Cytiva (1 mentions)
Quikchange lightning kit, by Agilent Technologies (1 mentions)
Immobilized metal affinity chromatography, by Bio-Rad (1 mentions)
E coli dh5α cells, by New England Biolabs (1 mentions)
Vivaspin column, by Sartorius (1 mentions)
12 protocols using t7 express cells
1
Purification of Bacterial RNA Polymerase
2
Propagation and Enumeration of T7 Phages
3
Xenopus Faf1 Protein Expression and Purification
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The X. laevis faf1 ORF was amplified from Xenopus cDNA using primers OK21/OK22 (Supplementary Table 1 ). The amplified faf1 product and pGEX-6p-1_GST-TEV-FLAG backbone derived from pGEX-6p-1_GST-TEV-FLAG-Ubxn7 were assembled in one construct using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). To generate the FLAG-Faf1UIM chimera, the pGEX-6p-1_GST-TEV-FLAG-Faf1 backbone was first amplified using primers OK85/OK86 (Supplementary Table 1 ) to delete the DNA encoding amino acids 241−368 of the Xenopus Faf1 sequence. The resulting PCR product was then assembled with the synthesized chimeric sequence containing DNA residues 919−984 of the Xenopus Ubxn7 cDNA inserted between DNA residues 879−882 of the Xenopus Faf1 cDNA using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). GST-TEV-FLAG-Faf1 and GST-TEV-FLAG-Faf1UIM were expressed in T7 Express cells (New England Biolabs) by induction with 0.5 mM IPTG overnight at 16 °C in LB media. FLAG-Faf1 and FLAG-Faf1UIM were then purified according to the FLAG-Ubxn7 purification protocol described above.
4
Plasmid Construction for Fluorescent Reporters
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The case studies made use of pUC19, pBAD24, pBAD/HisA, pACYC177, and pET28a plasmids as cloning vectors. All plasmids used and created are listed in Supporting Information: Table S1 and Appendix. The reporter genes for gfp and amcyan were obtained from plasmids pET‐GFP and pAmCyan (Takara Bio USA). Chimeric primers for each DNA insertion product and one reverse primer complementary to the pUC19 backbone were designed and are listed in Supporting Information: Table S2 . Chimeric primers contained two regions: (i) for amplifying the target DNA and (ii) homologous to the plasmid vector backbone to complete the cloning reaction, as shown in Figures 1 , 2 , 3 . All primers were synthesized by Integrated DNA Technologies (Coralville). Escherichia coli 10‐beta, E. coli BL21DE3, and T7 Express® cells (New England Biolabs; Ipswitch) were used for transformations.
5
Anaerobic Expression and Purification of IBSS and BSS
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All BSS and IBSS constructs were transformed into T7 Express cells (New England BioLabs) and a single colony was used to make a glycerol stock of each. Starter cultures were inoculated from glycerol stocks and grown overnight at 37°C at 220 rpm in LB containing either 50 μg/mL kanamycin and 100 μg/mL ampicillin for IBSSαγ and BSSαγ or 50 μg/mL kanamycin for IBSSβ and BSSβ. Expression cultures were inoculated with 10 mL of starter culture per 1 L of LB containing the corresponding antibiotics, 150 mg iron(II) ammonium sulfate hexahydrate (CAS: 783-85-9), and 47 mg L-cysteine. Expression, TALON purification, and concentration were carried out anaerobically as described above in “IbsAE large scale expression and purification .” For IBSSβ and BSSβ, the N-terminal His-tag was cleaved with His-tagged TEV protease at a ratio of 10:1 (β subunit:TEV protease, w/w). The reaction was gently mixed and left at 4°C for ∼24 hours (or until >80% completion as determined by SDS-PAGE) without agitation. The reaction mixture was purified on TALON resin as detailed above. Fractions containing pure IBSSβ or BSSβ, with the Histag removed, were pooled and buffer exchanged into 50 mM HEPES pH 8.0, 300 mM NaCl.
6
Expression and Purification of ACR4 and WOX5 Kinases
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Expression and purification was performed as described by Meyer et al [26 (link)]. Proteins were expressed for 5h in Rosetta2 (DE3) pLysS (Novagen) cells at 20°C with 1mM IPTG induction and the proteins purified as described (26). Briefly, pellets from 250 ml cultures were resuspended and lysed in 10 ml Lysis Buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 40 mM imidazole, 0.1% Triton X-100, 1 mM DTT, and 1 mM AEBSF). After centrifugation the supernatant lysate was incubated with Ni-NTA Superflow resin (Qiagen) and bound proteins eluted with Elution Buffer (50 mM Bis-Tris pH 7.2, 50 mM NaCl, 150 mM imidazole, 1 mM DTT) on ice. Protein quantities were determined by the Bradford method [27 (link)] and yields typically ranged from 1–5 mg of protein.
To isolate the MBP fused ACR4 kinase (mACR4), the vector was transformed into T7 Express cells (New England Biolabs) and plated on Ampicillin plates. Protein expression and purification was performed as described above, except Ampicillin (100 μg/ml) was used in cultures. Further purification of the IMAC enriched protein was performed by gel filtration on an FPLC system using the 10/300 Superdex G200 column (GE Healthcare) equilibrated with Column Buffer (50 mM Tris-HCl 7.4, 100 mM NaCl, 1 mM DTT). The MBP-fused WOX5 (mWOX5) protein was also expressed and purified as described above.
To isolate the MBP fused ACR4 kinase (mACR4), the vector was transformed into T7 Express cells (New England Biolabs) and plated on Ampicillin plates. Protein expression and purification was performed as described above, except Ampicillin (100 μg/ml) was used in cultures. Further purification of the IMAC enriched protein was performed by gel filtration on an FPLC system using the 10/300 Superdex G200 column (GE Healthcare) equilibrated with Column Buffer (50 mM Tris-HCl 7.4, 100 mM NaCl, 1 mM DTT). The MBP-fused WOX5 (mWOX5) protein was also expressed and purified as described above.
7
Purification of the MST2-SAV1 Complex
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DNA sequences corresponding to human MST2 (residues 1–484) and MBP‐tagged λ‐phosphatase were cloned into separate opening readings frames in pRSF‐Duet (Novagen). Nucleotides corresponding to human SAV1‐FL (residues 1–383) or a truncated variant, SAV1ΔN, (residues 196–383) were cloned into a modified pGEX‐2TK (Sigma‐Aldrich) encoding an N‐terminal His8‐StrepTagII‐mYFP tag.
T7 Express cells (New England Biolabs) were co‐transformed with both plasmids. Cells were grown in Terrific Broth to an OD600 of 2.0, protein expression induced by 0.25 mM IPTG and cells were additionally grown overnight at 16°C. Cells were lysed in 50 mM Tris pH 8.5, 400 mM NaCl and 10%(v/v) Glycerol. MST2:SAV1 complexes were purified by a combination of immobilised metal affinity chromatograph (IMAC; BioRad), streptactin affinity chromatography (Cytiva) and size‐exclusion chromatography (Superose 200; GE Healthcare). Complex was concentrated to approximately 10 μM in 10 mM Tris pH 8.5, 400 mM NaCl, 5% Glycerol and 5 mM βME and used immediately.
T7 Express cells (New England Biolabs) were co‐transformed with both plasmids. Cells were grown in Terrific Broth to an OD600 of 2.0, protein expression induced by 0.25 mM IPTG and cells were additionally grown overnight at 16°C. Cells were lysed in 50 mM Tris pH 8.5, 400 mM NaCl and 10%(v/v) Glycerol. MST2:SAV1 complexes were purified by a combination of immobilised metal affinity chromatograph (IMAC; BioRad), streptactin affinity chromatography (Cytiva) and size‐exclusion chromatography (Superose 200; GE Healthcare). Complex was concentrated to approximately 10 μM in 10 mM Tris pH 8.5, 400 mM NaCl, 5% Glycerol and 5 mM βME and used immediately.
8
Purification of Mycobacterial Arr Enzymes
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E. coli RNAP core and σ70 were purified exactly as described previously (19 (link)). M. smegmatis and M. abscessus Arr were expressed in T7 express cells (New England Biolabs) transformed with pET28 expression vector encoding N-terminal 6×His-tagged M. smegmatis Arr or M. abscessus Arr. Expression was induced with 0.4 mM isopropyl-β-d -thiogalactopyranoside (IPTG) in exponentially growing cells, which were then incubated overnight at room temperature on an orbital shaker (150 rpm). Cells were then harvested by centrifugation and resuspended in grinding buffer (50 mm Tris-HCl [pH 7.9], 10% glycerol, 200 mM NaCl, and protease inhibitor mixture [Roche]). Cells were then lysed by sonication and debris cleared by centrifugation. Arr enzymes were then purified by HisTrap HP (Cytiva) nickel affinity chromatography, concentrated, and dialyzed into storage buffer (50 mM Tris-HCl [pH 7.9], 50% glycerol, 200 mM NaCl, and 2 mM β-mercaptoethanol).
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9
Plasmid Mutagenesis and Transformation
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Mutations were made using the QuikChange Lightning Kit according to the manufacturer’s protocol (Agilent Technologies, Inc.). The PCR-generated plasmids were transformed into Escherichia coli NEB-5α cells by heat shock. The mutant plasmids were confirmed by sequencing and transformed into T7 Express cells (New England Biolabs).
10
Purification of RASSF5, MOB1A, and SARAH Domains
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