Plasmids used in this study were generated using standard polymerase chain reaction (PCR) techniques and/or type II and IIs restriction enzyme cloning. Restriction enzymes, Phusion DNA polymerase, T4 DNA Ligase, and Antarctic phosphatase were purchased from NEB. WT-ACE2 and Mut-ACE2 gene fragments were codon optimized and synthesized by Thermo Fisher and cloned into a pGIPZ backbone (Open Biosciences). The Spike protein from pcDNA3.1-SARS2-Spike was a gift from Fang Li (Addgene plasmid # 145032; http://n2t.net/addgene:145032 ; RRID:Addgene_145032);34 (link) this gene was cloned into a modified pcDNA 3.1 backbone (Clontech-Takara) with a beta-globin intron in the 5’ untranslated region for pseudotyping lentivirus. The Tet3G transactivator (pLVX-EF1a-TET3G) and cognate TRE3GV promoter (pLVX-TRE3G) (Takara) were cloned into modified pGIPZ and pLVX (Takara) backbones, respectively. In this context, the Spike protein (Addgene plasmid # 145032) was cloned downstream of the TRE3G promoter. All plasmids used in this study were sequence-verified, and maps will be published with the final manuscript. Chemically competent TOP10 Escherichia coli were used for transformation of all plasmids and subsequently grown at 37°C.
Protocol detail
Plasmid Generation for SARS-CoV-2 Studies
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Phusion DNA polymerase
T4 DNA Ligase
Antarctic phosphatase
pcDNA 3.1 backbone
pLVX-EF1a-TET3G
pLVX-TRE3G
Ace2
Backbones
Beta globin
Codon
Dna polymerase
Escherichia coli
Gene
Intron
Lentivirus
Maps
Phosphatase
Plasmids
Polymerase chain reaction
Restriction enzymes
Sars2 spike protein
Spike protein
T4 dna ligase
Transactivator
Untranslated region
Corresponding Organization : Northwestern University
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Variable analysis
independent variables
- WT-ACE2 and Mut-ACE2 gene fragments
- Spike protein from pcDNA3.1-SARS2-Spike
- Tet3G transactivator (pLVX-EF1a-TET3G) and cognate TRE3GV promoter (pLVX-TRE3G)
- Not explicitly mentioned
- Restriction enzymes, Phusion DNA polymerase, T4 DNA Ligase, and Antarctic phosphatase purchased from NEB
- Chemically competent TOP10 Escherichia coli used for transformation and growth at 37°C
- Positive control: Not specified
- Negative control: Not specified
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