Bacterial artificial chromosome (BAC) pBeloBAC11 (109 (link)), kindly provided by H. Shizuya (California Institute of Technology, Pasadena, CA), was used to assemble recombinant MERS-CoV infectious cDNA clones. This plasmid is a low-copy-number plasmid (one to two copies per cell) based on the Escherichia coli F factor (110 (link)) that allows the stable maintenance of large DNA fragments in bacteria. E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions. BAC plasmid and recombinant BACs were isolated and purified using a large-construct kit (Qiagen), following the manufacturer’s specifications.
Micropulser unit
Manufactured by Bio-Rad
Sourced in Spain
The MicroPulser unit is a compact and easy-to-use electroporation system designed for transforming bacterial and yeast cells. It delivers precise electrical pulses to facilitate the uptake of DNA, RNA, or other molecules into the target cells. The MicroPulser is a versatile laboratory instrument suitable for a wide range of electroporation applications.
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Lab products found in correlation
4 protocols using micropulser unit
1
Construction of Recombinant MERS-CoV Clones
2
Constructing Recombinant MERS-CoV Clones
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Bacterial artificial chromosome (BAC) pBeloBAC11 (62 (link)), provided by H. Shizuya, California Institute of Technology, Pasadena, CA, was used to assemble recombinant MERS-CoV and MERS-CoV mouse-adapted (MERS-MA30) infectious complementary DNA (cDNA) clones. This plasmid is a low–copy number plasmid (one, maximum two copies per cell) based on the Escherichia coli F factor (63 (link)) that allows the stable maintenance of large DNA fragments in bacteria. E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions. BAC plasmid and recombinant BACs were isolated and purified using a large-construct kit (Qiagen), following the manufacturer’s specifications.
Two plasmids were used for the expression of the MERS-CoV E protein: pcDNA3.1 for constitutive expression (Invitrogen; Thermo Fisher Scientific) (64 (link)) and TRE-Auto-rtTA-V10-2T for inducible expression provided by A. Das and B. Berkhout, Academisch Medisch Centrum Universiteit van Amsterdam, Amsterdam, The Netherlands (65 (link)).
Two plasmids were used for the expression of the MERS-CoV E protein: pcDNA3.1 for constitutive expression (Invitrogen; Thermo Fisher Scientific) (64 (link)) and TRE-Auto-rtTA-V10-2T for inducible expression provided by A. Das and B. Berkhout, Academisch Medisch Centrum Universiteit van Amsterdam, Amsterdam, The Netherlands (65 (link)).
3
Molecular Cloning and Expression of MERS-CoV Proteins
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E. coli DH10B (Gibco/BRL) cells were transformed by electroporation using a MicroPulser unit (Bio-Rad) according to the manufacturer’s instructions and grown on antibiotic-selective LB medium.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
MERS-CoV ORF4a-HA and ORF4b-HA nucleotide sequences were synthesized and cloned into pUC57 (GenScript). They were then PCR amplified, restriction digested and ligated into the pLKO plasmid. A pLKO ORF4b-3XFLAG plasmid was made by creating a PCR product replacing the HA tag with a 3XFLAG tag. This PCR product was restriction digested and ligated back into the pLKO plasmid. The resulting constructs were confirmed by restriction digest, PCR, and direct sequencing. The sMacro and GFP pcDNA3 plasmids were previously described [55 (link)]. The NSP15-3XFLAG plasmid was a generous gift of Michael Buchmeier (University of California-Irvine), and the KPNA-FLAG plasmids were a generous gift of Megan Shaw (Mount Sinai Medical Center). Cells were transfected using Polyjet (Amgen) or Lipofectamine 2000 (Fisher Scientific) as per the manufacturer’s instructions.
4
Assembly of ZIKV-RGN Infectious cDNA Clone
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The BAC plasmid pBeloBAC11 [40 (link)], kindly provided by H. Shizuya (California Institute of Technology, Pasadena, CA, USA), was used to assemble the ZIKV-RGN infectious cDNA clone. This plasmid is a synthetic low-copy-number plasmid (one copy per cell) based on the Escherichia coli (E. coli) F-factor [41 (link)] that minimize the toxicity problems in the bacteria of exogenous sequences. E. coli DH10B cells (Invitrogen, ThermoFisher Scientific) were used to amplify the BAC plasmids. Electrocompetent DH10B cells (Invitrogen, ThermoFisher Scientific) were transformed by electroporation using a MicroPulser unit (Bio-Rad, Madrid, Spain), according to the manufacturer’s instructions. BAC-based plasmids were isolated and purified using the Large-Construct kit (Qiagen, Hilden, Germany), following the manufacturer’s specifications.
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