According to the manufacturer’s instructions, to construct plasmids expressing the viral protein, DHAV-1 RNA was isolated using RNAiso Plus Reagent (TaKaRa). According to the manufacturer’s instructions, genomic DNA was then removed, and reverse transcription was performed using a PrimeScriptTM RT Reagent Kit (TaKaRa). VP0, VP1, 2A, 2B, 2C, 3AB, and 3D sequences were amplified from DHAV-1 cDNA with PCR and primers (Table 1 ). VP0, VP1, 2A, 2B, 2C, and 3D were integrated into the pCAGGs vector with a one-step cloning kit (Vazyme). 3AB was cloned into the pCMV-Myc vector with the DNA Ligation Kit (TaKaRa). The eukaryotic expression vector pCAGGs was gifted by Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The eukaryotic expression vector pCMV-Myc was purchased from TaKaRa. pCAGGs-VP3-Flag and pEGFP-N1-3C were also stocks in our laboratory (Lai et al., 2019 (link); Sun et al., 2019 (link)). Duck-derived G3BP1 gene was cloned into the pEGFP-C2 vector with the DNA Ligation Kit (TaKaRa). The pEGFP-C2 vector was purchased from YouBio.
Dna ligation kit
Manufactured by Takara Bio
Sourced in Japan, China, United States
The DNA Ligation Kit is a laboratory tool used to join DNA fragments together. It contains the necessary enzymes and reagents to facilitate the covalent bonding of DNA strands, a fundamental process in molecular biology and genetic engineering.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (7 mentions)
Primestar hs dna polymerase, by Takara Bio (6 mentions)
Restriction endonuclease, by Takara Bio (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Xhoi, by New England Biolabs (4 mentions)
Qiaprep spin miniprep kit, by Qiagen (4 mentions)
Pgl3 basic vector, by Promega (4 mentions)
Taq dna polymerase, by Takara Bio (3 mentions)
Wizard sv gel and pcr clean up system, by Promega (3 mentions)
Pgem t easy vector, by Promega (3 mentions)
Bamhi, by Toyobo (3 mentions)
Ab 3500 genetic analyzer, by Thermo Fisher Scientific (3 mentions)
In fusion hd cloning kit, by Takara Bio (3 mentions)
Restriction enzyme, by Takara Bio (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Ecori, by Toyobo (2 mentions)
Qiaquick gel extraction kit, by Qiagen (2 mentions)
Minibest plasmid purification kit, by Takara Bio (2 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Pezx fr01, by GeneCopoeia (2 mentions)
101 protocols using dna ligation kit
1
Plasmid Construction for DHAV-1 Viral Protein
2
Cloning and Sequencing of Human OR Genes
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All of the human OR genes were amplified from human genomic DNA (Promega, Madison, USA) using KOD FX (TOYOBO, Osaka, Japan) with sequence information from NCBI. Each forward primer included the EcoR I sequence followed by 25 to 40 base pairs of N-terminal sequence of each OR. Each reverse primer included the Spe I sequence followed by 25 to 40 base pairs of C-terminal sequence of each OR. The amplified OR genes and pME18S vectors containing a FLAG tag and the first 20 residues of bovine rhodopsin followed by EcoR I and Spe I sequence were restricted by EcoR I and Spe I(TAKARA BIO INC. Shiga, Japan) and ligated using DNA Ligation Kit (TAKARA). The sequences of cloned ORs were verified by sequencing. Some of the genes included several single nucleotide polymorphisms (SNPs) with sequence information from NCBI dbSNP (build141; http://www.ncbi.nlm.nih.gov/SNP/ ). Human receptor-transporting protein 1 short (RTP1S) gene was amplified from the human RTP1 gene (GE Healthcare UK Ltd., Buckinghamshire, England) using KOD FX (TOYOBO) with sequence information from NCBI. Forward and reverse primer included EcoR I and Xho I sequence followed by N-terminal or C-terminal sequence of RTP1S, respectively. The amplified RTP1S gene and pME18S vector were restricted by EcoR I and Xho I (TAKARA) and ligated using DNA Ligation Kit (TAKARA). The sequences of cloned RTP1S were verified by sequencing.
3
Generation and Validation of APOBEC3C Overexpressing Cell Lines
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Wild-type APOBEC3C consensus coding sequence (CCDS) (APOBEC3C-pENTER vector, WZ Biosciences) was amplified by PCR (forward primer: 5’-CCGTCAGATCCGCTAGTAATAC-3’, reverse primer: 5’-CCGGAATTCTGTGGTATGGCTGATTATGATC-3’). Then, BamHI and EcoRI were employed to generate sticky ends. The pCDH-CMV-puro plasmid was digested with the same enzymes, and restriction enzyme-digested products were purified by electrophoresis and Universal DNA Purification Kits (Cat#DP214-03, Tiangen, China). The CCDS fragment and pCDH plasmid were ligated using DNA Ligation Kits (Cat# 6021, Takara, Japan) and then transformed into DH5α cells for further selection and amplification.
EndoFree Mini Plasmid Kit II (Cat# DP118-02, Tiangen) was used to extract pCDH-APOBEC3C (pCDH-A3C). Then, 7.5 µg pCDH-A3C or pCDH backbone plus 5.625 µg psPAX2 and 1.875 µg pMD2.G were incubated with 30 µL Hieff Trans Liposomal Transfection Reagent (Cat# 40802ES03, Yeason, China) in 1 mL DMEM. The mixture was added to 293T cells to generate lentiviruses, which were used to transfect pancreatic cell lines. Puromycin was added to culture mediums (4 µg/mL) for selection. Four months after transfection, total DNA was extracted from A3C-overexpressing cell lines and subjected to Sanger sequencing to verify that A3C sequence in the plasmid did not undergo genetic modifications.
EndoFree Mini Plasmid Kit II (Cat# DP118-02, Tiangen) was used to extract pCDH-APOBEC3C (pCDH-A3C). Then, 7.5 µg pCDH-A3C or pCDH backbone plus 5.625 µg psPAX2 and 1.875 µg pMD2.G were incubated with 30 µL Hieff Trans Liposomal Transfection Reagent (Cat# 40802ES03, Yeason, China) in 1 mL DMEM. The mixture was added to 293T cells to generate lentiviruses, which were used to transfect pancreatic cell lines. Puromycin was added to culture mediums (4 µg/mL) for selection. Four months after transfection, total DNA was extracted from A3C-overexpressing cell lines and subjected to Sanger sequencing to verify that A3C sequence in the plasmid did not undergo genetic modifications.
4
High-Fidelity DNA Polymerase Reaction Protocols
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All chemicals were purchased from Nacalai Tesque (Tokyo, Japan) or Wako Pure Chemical (Tokyo, Japan) unless otherwise specified. KOD Plus high-fidelity DNA polymerase was purchased from Toyobo (Tokyo, Japan). Restriction enzymes and the DNA ligation kits used were from Takara (Shiga, Japan).
5
Optimized Lip1 Gene Expression in Pichia pastoris
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The Lip1 gene sequence was optimized and constructed based on the protein-coding sequence of C. rugosa Lip1 (GenBank accession number: X64703.1)6 (link) (ESI DOC S1† ). Escherichia coli DH5α was maintained at 37 °C in Luria-Bertani medium for gene cloning. The vector pPICZαA (Novagen, Darmstadt, Germany) and Pichia pastoris GS115 were used for gene expression. All P. pastoris transformants were cultured in YPD medium (1% yeast extract, 2% tryptone, and 2% dextrose), BMGY medium (2% tryptone, 1% yeast extract, 50 mM potassium phosphate, pH 7.5, 1.34% yeast nitrogen base, 4 × 10−5% biotin, 1% glycerol) and BMMY medium (2% tryptone, 1% yeast extract, 50 mM potassium phosphate, pH 7.5, 1.34% yeast nitrogen base, 4 × 10−5% biotin, 1% methanol) containing 100 μg mL−1 zeocin (Invitrogen, Carlsbad, CA, USA) at 28 °C. Restriction endonucleases, Taq DNA polymerase, DNA Ligation Kits and PrimeSTAR™ HS DNA Polymerase were purchased from TaKaRa (Otsu, Japan). Kits for plasmid extraction, and gel extraction were bought from Omega Bio-tek (Norcross, GA, USA). p-Nitrophenyl (p-NP) esters were commercially obtained from Sigma-Aldrich (USA). All other chemicals were got from Sino-pharm Chemical Reagent Co., Ltd (Shanghai, China).
6
Bacterial Strain and Plasmid Propagation
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Details of bacterial strains and plasmids used in this study are in Table 1 . Propagation of P. aeruginosa strains was in liquid or solid LB or M9 minimal (1.5% agar) medium (0.6% Na2HPO4, 0.3% KH2PO4, 0.1% NH4Cl, 0.05% NaCl, 3 × 10−4% CaCl2, 1 mL 1 mol/L MgSO4·7H2O, 10 mL 20% glucose). Escherichia coli strains were propagated in LB medium (1.5% agar). The antibiotics used were kanamycin (50 μg/mL) and carbenicillin (100 μg/mL) for P. aeruginosa, and ampicillin (100 μg/mL) and tetracycline (25 μg/mL) for E. coli. PrimeSTAR HS DNA polymerase, DNA ligation kits and restriction enzymes were purchased from TaKaRa Biotechnology (Dalian, China). Kits for extracting genomic DNA, DNA gel and preparing plasmids (Omega Bio-Tek, Doraville, GA, USA) were used according to the manufacturer's instructions. DNA fragments were sequenced by Shanghai Sunny Biotechnology (Shanghai, China). Primers were synthesized by Wuhan Anygene Biological Technology (Wuhan, China). Other regular methods were completed according to defined protocols.
7
Quantum Chemical Calculations for Enzyme Engineering
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The computational designs were run on an Intel® Xeon® Processor X5690 processor (12M Cache, 3.46 GHz, 6.40 GT/s Intel® QPI) using the CentOS 6.4 operating system. We would like to thank our collaborators at Jilin University and Huazhong Normal University for providing the resources needed for quantum chemical calculations. The Rosetta package was downloaded from http://www.rosettacommons.org .
Restriction endonuclease, Taq DNA polymerase, DNA ligation kits and PrimeSTAR® HS DNA polymerase were purchased from TaKaRa (Otsu, Japan). Kits for plasmid and gel extractions were purchased from Omega Bio-tek (Norcross, GA, USA). p-Nitrophenyl (p-NP) ester was purchased from Sigma-Aldrich (St. Louis, MO, USA). Reduced glutathione and oxidized glutathione were purchased from Merck (Darmstadt, Germany). Protein marker was purchased from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were purchased from China Pharmaceutical Chemical Reagent Co., Ltd. (Shanghai, China).
Restriction endonuclease, Taq DNA polymerase, DNA ligation kits and PrimeSTAR® HS DNA polymerase were purchased from TaKaRa (Otsu, Japan). Kits for plasmid and gel extractions were purchased from Omega Bio-tek (Norcross, GA, USA). p-Nitrophenyl (p-NP) ester was purchased from Sigma-Aldrich (St. Louis, MO, USA). Reduced glutathione and oxidized glutathione were purchased from Merck (Darmstadt, Germany). Protein marker was purchased from ThermoFisher Scientific (Waltham, MA, USA). All other chemicals were purchased from China Pharmaceutical Chemical Reagent Co., Ltd. (Shanghai, China).
8
Recombinant Protein Expression in CHO Cells
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The pcDNA3 mammalian expressing vector, Chinese hamster ovary suspension (CHO-S)
cells, FreeStyle MAX reagent, and FreeStyle CHO expression medium, were
purchased from Invitrogen (San Diego, CA, USA). CHO-K1 cells were obtained from
the Korean Cell Bank (KCB) (Seoul, Korea). The polymerase chain reaction (PCR)
reagents, restriction enzymes, and DNA ligation kits were obtained from Takara
(Shiga, Japan). Ham’s F-12 medium, Opti-MEM I, serum-free CHO-S-SFM II,
and Lipofectamine 2000 were purchased from Gibco BRL (Grand Island, NY, USA).
Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT,
USA). Disposable spinner flasks and glass flasks were obtained from Corning
(Corning, NY, USA). The cyclic adenosine monophosphate (cAMP) Dynamic 2
immunoassay kit was purchased from Cisbio Bioassay (Codolet, France). PSMG
enzyme-linked immunosorbent assay (ELISA) kit was purchased from DRG
International (Mountainside, NJ, USA). The QIAprep-Spin plasmid kit was
purchased from Qiagen (Hilden, Germany). The oligonucleotides were synthesized
by Genotech (Daejeon, Korea). All other reagents were obtained from
Sigma-Aldrich (St. Louis, MO, USA).
cells, FreeStyle MAX reagent, and FreeStyle CHO expression medium, were
purchased from Invitrogen (San Diego, CA, USA). CHO-K1 cells were obtained from
the Korean Cell Bank (KCB) (Seoul, Korea). The polymerase chain reaction (PCR)
reagents, restriction enzymes, and DNA ligation kits were obtained from Takara
(Shiga, Japan). Ham’s F-12 medium, Opti-MEM I, serum-free CHO-S-SFM II,
and Lipofectamine 2000 were purchased from Gibco BRL (Grand Island, NY, USA).
Fetal bovine serum (FBS) was obtained from Hyclone Laboratories (Logan, UT,
USA). Disposable spinner flasks and glass flasks were obtained from Corning
(Corning, NY, USA). The cyclic adenosine monophosphate (cAMP) Dynamic 2
immunoassay kit was purchased from Cisbio Bioassay (Codolet, France). PSMG
enzyme-linked immunosorbent assay (ELISA) kit was purchased from DRG
International (Mountainside, NJ, USA). The QIAprep-Spin plasmid kit was
purchased from Qiagen (Hilden, Germany). The oligonucleotides were synthesized
by Genotech (Daejeon, Korea). All other reagents were obtained from
Sigma-Aldrich (St. Louis, MO, USA).
9
Cloning and Expression of C. parvum Proteins
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3.0 × 108C. parvum, strain HNJ-1, sporozoites were used for DNA extraction. The sporozoite genomic DNA was extracted by using the QIAamp DNA Micro Kit (QIAGEN, Venlo, Netherlands) according to the manufacturer’s instructions. CpEF1α open reading frame was amplified by PCR with a pair of primers [forward, CpEF1-α-EcoRI-F (5’-ACT GAA TTC ATG GGT AAG GAA AAG ACT C-3’), and reverse, CpEF1α-NotI-R (5’-ACT GCG GCC GCT TAC TTC TTC TTG GAA GTG G-3)] that provided EcoRI and NotI restriction sites. The C. parvum actin (CpActin) open reading frame was amplified by PCR with a pair of primers [forward, CpActin-SalI-F (5’-ACT GGT CGA CTC ATG AGT GAA GAAGAA ACA C-3’), and reverse, CpActin-NotI-R (5’-ACT GCG GCC GCT TAG AAG CAC TTT CTG TG-3)] that provided SalI and NotI restriction sites. The amplified fragments of CpEF1α, and CpActin were digested with EcoRI/NotI and SalI/NotI, respectively, and were cloned into the pGEX-6P-1 vector (GE Healthcare UK Ltd, Buckinghamshire England) by using a DNA ligation kit (TAKARA BIO INC., Shiga, Japan) to express a glutathione S-transferase (GST) fusion protein.
10
Genome DNA Extraction and Sequencing
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Genome DNA was extracted from A431 cells (wild type, clones #1 and #2) using GeneArt Genomic Cleavage Detection Kit (Thermo Fisher Scientific), following the manufacturer’s protocol. The sequence around the target of CRISPR/Cas9 was amplified using the following pair of primers: Piezo1 gDNA forward and Piezo1 gDNA reverse (Table S5 ). The amplified product was purified using Wizard SV Gel and PCR Clean-up system (Promega) and inserted into T-Vector pMD20 (Takara Bio) using DNA Ligation Kit (Takara Bio) according to the manufacturer’s protocol. The ligation mixture was introduced into Escherichia coli DH5α and the insert sequence was verified by standard sequencing.
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