CPPs-SOD1 expression vectors were constructed in our laboratory. CPPs-SOD1 genes were cloned from HK-2 cDNA using PCR. The sequences of the forward and reverse primers are listed in Table S1 . Forward primers contain an NcoI restriction site, a sequence of CPPs, and PET-15b. Reverse primers contain an XhoI restriction site, sequences of six histidines, and PET-15b. Then, the CPPs-SOD1 gene was fused to NcoI-XhoI-digested pET15b by exnase (Vazyme, China). The sequencing results are shown in Figure S1 .
Exnase
Manufactured by Vazyme
Sourced in China
Exnase is an enzyme-based reagent designed for nucleic acid extraction. It functions to effectively isolate and purify DNA or RNA from various biological samples.
Automatically generated - may contain errors
6 protocols using exnase
1
Constructing CPPs-SOD1 Expression Vectors
2
Cloning and Characterization of PPARγ Truncations
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DNA fragments encoding the proteins of interest were synthesized by GenScript Biotech Corporation (Nanjing, China) and amplified by PCR with Phanta® Max Super-Fidelity DNA Polymerase (Vazyme, P505-d1), while the coding region for PPARγ truncations (residues 1–39, residues 31–108, residues 138–221, residues 1–221, residues 31–221, residues 138–504 and residues 237–504) were generated by PCR from a plasmid containing full-length PPARγ with appropriate sets of primers. Exnase (Vazyme, C214-02-AF) was used to insert these sequences into the pET-28a vector containing an mCherry or mEGFP tag. Plasmid inserts were confirmed by BioSune Sanger sequencing, reading from both ends of the insert. For the construction of sgRNA expression plasmids, oligos were custom synthesized, annealed and cloned into pGL3-U6-sgRNA-EGFP vector (Addgene, 107721).
3
SARS-CoV-2 N Protein Expression Constructs
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SARS-CoV-2 N protein-coding fragments were polymerase chain reaction (PCR)-amplified from the N protein expression construct in the PUC57 vector (Tsingke Biotech, Hangzhou, China) and subcloned into the pCS2 vector, while the coding region for SARS-CoV-2 N protein deletions (residues 1–175, residues 1–210, residues 51–C, residues 161–C, residues 211–C, and ΔSR) were amplified by PCR from a plasmid containing full-length SARS-CoV-2 N protein with appropriate sets of primers and was confirmed by sequencing. DNA fragments encoding human ubiquitin-specific peptidase 10 (USP10), double-stranded RNA-dependent protein kinase (PKR), and G3BP2 were PCR-amplified from cDNA that was reverse transcribed from HEK293T total RNA with reverse transcriptase (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Exnase (Vazyme, Nanjing, China) was used to insert these sequences into the CMV-Flag or CMV-Myc backbone.
4
Heat-Induced GUS Expression Analysis
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Various TaMBF1c‐7B promoter‐driven β‐glucuronidase (GUS) reporters (P1:GUS, P2:GUS and P3:GUS) were constructed by recombining the PCR‐amplified DNA fragments (200, 525 and 1500 bp) upstream of GUS in the pCAMBIA1300 vector using Exnase (Vazyme). The GUS reporters and an internal control (35S:RFP) were cointroduced into the N. benthamiana leaf epidermal cells via Agrobacterium tumefaciens strain GV3101 (Liu et al., 2010 (link)), and the empty pCAMBIA1300 vector (P0:GUS) was used as a negative control. Plants were kept at 22°C for 3 d before being induced by heat stress (38°C for 1 h). Infiltrated leaves before and after heat treatment were harvested and used for GUS expression analysis. MAS:TaHsfA constructs for transactivation assays were generated by cloning the coding sequences of 33 TaHsfA genes (Table S3 ) from CB037 into the p‐super1300 vector through recombination ligation, respectively. A mixture of an effector (MAS:TaHsfA), a reporter (P3:GUS) and an internal control (35S:RFP) was cotransfected into N. benthamiana as described above, and the empty p‐super1300 vector was used as a negative control. Three days after infiltration, the infected leaves were harvested and used for GUS expression analysis. GUS activity in all samples was normalized against the red fluorescent protein (RFP) activity.
5
Expression and Purification of MtrA Protein
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A fragment encoding MtrA (Rv3246c) was amplified from the M. tuberculosis H37Rv genomic DNA using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The primers (5′-cagcaaatgggtcgcggatccATGGACACCATGAGGCAAAGG-3′), (5′-gtggtggtggtggtgctcgagTCACGGAGGTCCGGCCTT-3′) were designed using Primer3 (https://www.primer3plus.com , accessed on 23 November 2020) and synthesized by Tsingke Biotechnology (Beijing, China). The purified fragment was cloned into pET28a and linearized by BamH I and Xho I (NEB, England) double-digestion using Exnase (Vazyme, Nanjing, China).
E. coli BL21 (DE3) transformed with pET28a-MtrA was induced with 0.5 mM IPTG at 16 °C for 20 h when OD600 reached 0.6. For protein purification following routine procedures, a 250 mM final concentration of Imidazole was used for elution after being loaded on a Ni-NTA 6FF prepacking chromatographic column (Sangon Biotech, Shanghai, China), and eluted MtrA was desalted and concentrated using a 10 Kda protein concentrator (Millipore, MA, USA) in storage buffer (20 mM HEPES, 100 mM NaCl and 5% glycerol pH 7.5). The purified protein was stored at −80 °C for further experiments after the purity and concentrations were assayed by 12% SDS-PAGE (Yeasen Biotechnology, Shanghai, China) and a BCA kit (Sangon Biotech, Shanghai, China).
E. coli BL21 (DE3) transformed with pET28a-MtrA was induced with 0.5 mM IPTG at 16 °C for 20 h when OD600 reached 0.6. For protein purification following routine procedures, a 250 mM final concentration of Imidazole was used for elution after being loaded on a Ni-NTA 6FF prepacking chromatographic column (Sangon Biotech, Shanghai, China), and eluted MtrA was desalted and concentrated using a 10 Kda protein concentrator (Millipore, MA, USA) in storage buffer (20 mM HEPES, 100 mM NaCl and 5% glycerol pH 7.5). The purified protein was stored at −80 °C for further experiments after the purity and concentrations were assayed by 12% SDS-PAGE (Yeasen Biotechnology, Shanghai, China) and a BCA kit (Sangon Biotech, Shanghai, China).
6
Promoter Activity Quantification in Arabidopsis
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Two different promoter sequences were cloned with speci c primers using JKK378 and HXW as template. The puri ed and recovered promoter sequence fragments were assembled into pGreen-0800 vector using Exnase® (ClonExpress® One Step Cloning Kit, Vazyme, Nanjing, China). The vector included Renilla luciferase (REN) initiated by 35S and luciferase (LUC) started by the target promoter. The recombined vector transformed into E. coli DH5α. Correct sequencing of positive plasmids transformed Arabidopsis protoplasts with PEGmediated method (Lu et al. 2020) (link). In this study, the active and extended leaves of Columbia wild-type Arabidopsis were selected for the preparation of protoplasm. Fluorescence detection was performed 18 hours after conversion using microplate reader (Biotek Synergy H1, USA). Promoter activity was measured by the ratio of LUC divided by REN.
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