The strain TMCΔT: BL21-AI (Life Technologies, Carlsbad, CA, USA) ΔdinB, ΔumuDC, ΔrecA, was used for protein purification, and was constructed by P1 transduction (Thomason et al., 2007 (link)) using as a host the BL21-AI ΔdinB, ΔumuDC strain (Cafarelli et al., 2013 (link)). The construction of the RecA overproducing plasmid (pILRecA) is described below while the DinB overproducing plasmid (pDFJ1) has been previously published (Jarosz et al., 2006 (link)). The TMCΔT strain with the overproducing plasmids was grown in Luria broth medium with ampicillin [TMCΔT/pDFJ1 (Jarosz et al., 2006 (link)); 100 μg/mL] or kanamycin (TMCΔT/pILRecA; 35 μg/mL). Protein induction conditions are described below. All oligonucleotides used in this work are listed in Supplementary Table 1 .
Bl21 ai
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan
BL21-AI is a competent E. coli strain used for protein expression. It is engineered to provide tight control of protein expression under the regulation of the araBAD promoter.
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Lab products found in correlation
Pdest17, by Thermo Fisher Scientific (3 mentions)
Pdest15 vector, by Thermo Fisher Scientific (2 mentions)
Coomassie brilliant blue stain, by Bio-Rad (2 mentions)
Bl21 de3, by Thermo Fisher Scientific (2 mentions)
Pentr d topo, by Thermo Fisher Scientific (2 mentions)
L arabinose, by Merck Group (2 mentions)
Pdest17 vector, by Thermo Fisher Scientific (2 mentions)
Pdest14, by Thermo Fisher Scientific (2 mentions)
Histrap nickel affinity column, by GE Healthcare (1 mentions)
Akta system, by GE Healthcare (1 mentions)
Mono q column, by GE Healthcare (1 mentions)
Pet15b, by Merck Group (1 mentions)
Pcoladuet, by Merck Group (1 mentions)
Pcdfduet, by Merck Group (1 mentions)
Pacycduet, by Merck Group (1 mentions)
Pdest15, by Thermo Fisher Scientific (1 mentions)
Pierce glutathione magnetic agarose beads, by Thermo Fisher Scientific (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
Hitrap desalting column, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
41 protocols using bl21 ai
1
TMCΔT: Modified BL21-AI Strain Construction
2
Purification and Visualization of Shank3 Fragments
3
Purification of His6-tagged YtfB Protein
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His6-YtfB was produced and purified from the overproduction E. coli strain BL21 AI (Invitrogen). Strains were grown overnight in LB containing ampicillin before being diluted in fresh media to a starting optical density (OD600) of 0.05. Cultures were incubated at 37 °C with shaking until the OD600 reached 0.4. Arabinose was added to a final concentration of 0.2% to induce protein expression, and cultures were incubated for a further 4 h before cells were harvested by centrifugation. The pellet was resuspended in lysis buffer (25 mM Tris pH 7.6, 2 mM DTT, 1 mg/ml lysozyme, 50 µg/ml DNAse, Roche complete protease tablet), and incubated at 25 °C for 1 h. Cell lysis was performed by freeze/thaw in liquid nitrogen, and the sample was sonicated and centrifuged at high speed for 30 min 4 °C. Supernatant containing soluble protein was filtered using a 0.2 µm syringe followed by protein capture using a Nickel Affinity HisTrap column and AKTA system (GE Healthcare). Anion ion exchange chromatography was performed to increase purity of the protein using a MonoQ column (GE Healthcare). His6-YtfB was dialysed into buffer containing 20 mM TrispH 8.0, 0.2 M NaCl, 10% glycerol. Concentration was calculated by measuring UV absorbance at 280 nm. Protein purity was determined to be ~95% based on visual judgement of Coomassie brilliant blue stained protein separated by SDS-PAGE.
4
Construction of Synthetic RNA Circuits in E. coli
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The following E. coli strains were used in this study: BL21 DE3 (Invitrogen; F− ompT hsdSB (rB− mB−) gal dcm), BL21 AI (Invitrogen; F− ompT hsdSB (rB− mB−) gal dcm araB::T7RNAP-tetA), and DH5α (Invitrogen; endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK− mK+) λ−).
The backbones for the plasmids used in this research were taken from the commercial vectors pET15b, pCDFDuet, pCOLADuet, and pACYCDuet (EMD Millipore). The switch RNA of the NIMPLY complex was constructed using ACTS Type II N3 and ACTS Type II N7 from previous research [46 (link)] and was constructed in pACYCDuet. All the trigger RNAs and trigger cassettes were constructed in pCDFDuet. All the antisense RNAs and antisense cassettes were constructed in pET15b. The switch RNAs of the AND gate and the NIMPLY gate were constructed in pCOLADuet. All constructs were cloned via blunt end ligation [94 (link)], Gibson Assembly [95 (link)], circular polymerase extension cloning (CPEC) [96 (link)], and/or round-the-horn site-directed mutagenesis [97 (link)]. The plasmid architecture and specific part sequences are listed inTables S3–S11 . Plasmids were constructed in E. coli DH5α and purified using the EZ-PureTM plasmid Prep Kit. Ver. 2 (Enzynomics). Plasmid sequences were confirmed by DNA sequencing after every cloning step. Plasmids were transformed through chemical transformation [98 (link)].
The backbones for the plasmids used in this research were taken from the commercial vectors pET15b, pCDFDuet, pCOLADuet, and pACYCDuet (EMD Millipore). The switch RNA of the NIMPLY complex was constructed using ACTS Type II N3 and ACTS Type II N7 from previous research [46 (link)] and was constructed in pACYCDuet. All the trigger RNAs and trigger cassettes were constructed in pCDFDuet. All the antisense RNAs and antisense cassettes were constructed in pET15b. The switch RNAs of the AND gate and the NIMPLY gate were constructed in pCOLADuet. All constructs were cloned via blunt end ligation [94 (link)], Gibson Assembly [95 (link)], circular polymerase extension cloning (CPEC) [96 (link)], and/or round-the-horn site-directed mutagenesis [97 (link)]. The plasmid architecture and specific part sequences are listed in
5
Soluble Expression of FlhF Protein
6
Overexpression and Purification of LacY in E. coli
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LacY was overexpressed and purified in E. coli as previously described30 (link). Briefly, BL21-AI (Invitrogen) cells were transformed with the lacy gene cloned in the pET28 plasmid (Novagen) with a C-terminal 10-histidine tag, grown in LB media to an OD600 of 0.8 then expression was induced with 1 mM IPTG and 0.1% (w/v) arabinose and growth continued until saturation. The cells were harvested, passed through a microfluidiser (Constant Systems) and the membranes isolated by centrifugation for 30 mins at 100,000 × g. Membrane proteins were solubilised for 2 hours in 50 mM sodium phosphate, 100 mM NaCl, 10% (v/v) glycerol, 20 mM imidazole, 2 mM B-mercaptoethanol, pH 7.4 with 2% (w/v) DDM. Solubilised LacY was bound to a 1ml Histrap column (GE Healthcare) and washed with buffer containing 75 mM imidazole and 0.05% (w/v) DDM. Purified protein was eluted with 500 mM imidazole and exchanged into a final buffer of 50 mM sodium phosphate, 10% (v/v) glycerol, 1 mM B-mercaptoethanol, 0.05% DDM pH 7.4 on a 5 ml Hitrap desalting column (GE Healthcare).
7
Expression of Non-Fused Protein Complex for SAXS and SANS
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For SAXS measurements, the non-fused protein complex was co-expressed in E. coli strain BL21-AI (Invitrogen, Carlsbad, CA). The culture was grown at 37℃ in TB media with 70 mM of Na/K-Pi (pH 6.7) and 50 mg/L of kanamycin. Expression was induced with the mixture of 0.1% arabinose and 2 mM IPTG, at OD600 = 1.6–2.0. Simultaneously, all-trans retinal solution in ethanol was added to a final concentration 10 µM, and cells were further cultivated 4 h at 37 °C. For SANS measurements, the non-fused protein complex was co-expressed in E. coli strain BL21(DE3). The culture was grown in TB-5052 media60 (link) with 100 mM of Na/K-Pi (pH 6.7), 25 mM of ammonium sulfate and 100 mg/L of kanamycin. The cells were incubated at 37 °C until OD600 reached 1.0–1.2, when all-trans retinal solution in ethanol was added to final concentration 10 µM, and cells were further cultivated overnight at 20 °C as was described in61 (link) for expression of the NpSRII.
8
Preabsorption of Bovine Antibodies
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Cattle sera were incubated at 37 °C for 2 h in pre-adsorption solution to avoid unspecific binding of bovine antibodies [13 (link)]. The solution was 5% (w/v) non-fat dried milk and 100 μg/mL of bacterial culture lysate supernatant in 0.5% (v/v)-solution of Tween 20 in 1× PBS. Briefly, the bacterial lysate was obtained from 50 mL of overnight Escherichia coli culture of strain BL21 AI™ (Invitrogen). Then, the culture was harvested and centrifuged at 17,257 g during 10 min at 4 °C. The supernatant was discarded and the pellet resuspended with gentle agitation for 2 h at 4 °C in 4 mL of lysis buffer (100 mM Tris HCl, pH: 7.5; 500 mM NaCl; 20% glycerol; 1% Triton X-100; 20 mM imidazole (pH: 7.4); plus 1 mg/mL Lysozyme and 0.5 mM phenylmethylsulfonyl fluoride). The suspension was sonicated by three cycles (1 min/cycle) and centrifuged at 14,501 g during 30 min at 4 °C. The supernatant was separated from the pellet and used. Total protein was quantified with a BCA commercial kit (Pierce, Rockford, IL, USA) and stored at −20 °C until used.
9
Recombinant Expression of FIPV Proteins
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The regions encoding the E protein and the S1 domain of the S protein of FIPV serotype I strain KU-2 and the E protein of the FIPV serotype II strain 79-1146 were amplified by RT-PCR using the method described by Takano et al. [24 (link)]. The primers used to amplify each region are shown in Table 1 . The PCR products were inserted into pENTR/D-TOPO (Invitrogen, USA), and then into pDEST15, using recombination. This construct was then used to transfect E. coli strain BL21-AI (Invitrogen, USA). Bacterial cultures were grown for 2-3 h at 37 °C to an OD600 of 0.4, and the expression of proteins was induced by the addition of 0.2 % (w/v) L-arabinose (Sigma Aldrich, USA). We normalized GST+E protein and GST+S1 protein expression.
Sequences of primers used in this study
| Orientation | Nucleotide sequence | Length (bp) | |
|---|---|---|---|
| FIPV KU-2 E | Forward | 5′-CACCATGATGTTTCCTAGGGCA-3′ | 246 |
| Reverse | 5′-TCAAACCAAGAGTGCTTCGTT-3′ | ||
| FIPV 79-1146 E | Forward | 5′-CACCATGACGTTCCCTAGGGCATTTAC-3′ | 246 |
| Reverse | 5′-TCAAACCAAAAATGCTTCGTCGGGA-3′ | ||
| FIPV KU-2 S1 | Forward | 5′- CACCGTCACTGATTTCAGCCTGC-3′ | 932 |
| Reverse | 5′- TTATTCATCTTTACCAAAACAGAGC-3′ |
10
Producing Antibodies for Studying Telomere Proteins
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To produce an antibody specific for OsGSL5 of 1910 amino acids (aa), the cDNA sequence encoding the 1,009–1,260 aa position was amplified using the above cDNA library as a template (Supplemental Figure S5 and Supplemental Table S2 ), and cloned into pDEST17 vectors (Invitrogen). The His-tagged protein expressed in Escherichia coli strain BL21-AI (Invitrogen) was purified using Ni-NTA agarose resin (FUJIFILM) and immunized to rabbits and guinea pigs.
To observe telomere behaviors in PMCs, the antibody was raised against rice PROTECTION OF TELOMERE1 (OsPOT1), which is encoded by a single-gene locus Os04g0467800 (LOC_Os04g39280.1), while the Arabidopsis (A. thaliana) genome has two putatively paralogous loci, POT1a and POT1b (Shakirov et al., 2005 (link)). Procedures to raise antisera are the same as above.
To observe telomere behaviors in PMCs, the antibody was raised against rice PROTECTION OF TELOMERE1 (OsPOT1), which is encoded by a single-gene locus Os04g0467800 (LOC_Os04g39280.1), while the Arabidopsis (A. thaliana) genome has two putatively paralogous loci, POT1a and POT1b (Shakirov et al., 2005 (link)). Procedures to raise antisera are the same as above.
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