Three recombinant vectors, pPIC9K-Aoxyn11AG21I, -Aoxyn11AY13F and -Aoxyn11AG21I–Y13F, were linearized with SalI, and transformed into P. pastoris GS115, respectively, by electroporation using the Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA). All P. pastoris transformants were primarily screened based on their abilities to grow on the MD plate, and then successively inoculated on the YPD plates containing G418 at concentrations of 1.0, 2.0 and 4.0 mg mL−1 for the screening of multiple copies of the integrated target gene. The P. pastoris transformant resisting high geneticin G418 concentration may have multiple copies of a gene, which can lead to the high expression of recombinant protein/enzyme (Invitrogen, USA). However, the expression level was not directly proportional to G418 concentration (Zhang et al. 2014 (link)). Thereby, a total of 60 P. pastoris transformants resistant to G418 of 4.0 mg mL−1, separately containing Aoxyn11AG21I, Aoxyn11AY13F and Aoxyn11AG21I–Y13F, were picked out for expression tests.
Gene pulser apparatus
Manufactured by Bio-Rad
Sourced in United States
The Gene Pulser apparatus is a laboratory instrument designed to deliver controlled electric pulses to cells, facilitating the introduction of DNA, RNA, or other molecules into the cell's interior. The device generates and delivers precisely controlled electric field pulses, which temporarily permeabilize the cell membrane, allowing the desired molecules to enter the cell.
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Lab products found in correlation
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (5 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Roche (2 mentions)
Prep plasmid midi kit, by Qiagen (2 mentions)
Pulse controller, by Bio-Rad (2 mentions)
S1 nuclease, by Takara Bio (2 mentions)
Histrap hp column, by GE Healthcare (2 mentions)
Ktaprime plus system, by GE Healthcare (2 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
Pfx dna polymerase, by Thermo Fisher Scientific (1 mentions)
Dna sequencing, by GenScript (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Multi copy pichia expression kit, by Thermo Fisher Scientific (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Oleic acid albumin dextrose catalase oadc, by BD (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
68 protocols using gene pulser apparatus
1
Screening Recombinant P. pastoris Transformants
2
Detecting Gh-LYK1/2 Dimerization in Tobacco Leaves
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To detect the dimerization of Gh-LYK1/2 in tobacco leaves, the full length ORFs of Gh-LYK1 and Gh-LYK2 were cloned into p2YN and p2YC, respectively. The resulting plasmids were individually introduced into A. tumefaciens strain EHA105 by electroporation using a Gene Pulser Apparatus (Bio-Rad, Hercules, CA) as described (Huang et al., 2009 (link)). The bimolecular fluorescence complementation (BiFC) assay was then performed as described previously (Yang et al., 2007 (link)). To enhance the ectopic expression, Agrobacterium harboring the gene silencing suppressor P19 was co-infiltrated into tobacco leaves. Emission of YFP fluorescence was detected and the cells were imaged under a confocal microscope (LSM 780, Germany) at 48 h post infiltration.
3
In Vitro Amplification of Virus
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RNA transcript was in vitro transcribed by the mMESSAGE mMACHINE T7 Transcription Kit (ThermoFisher Scientific) in 30μl system with some modifications. Twenty micrograms of viral RNA and 20μg N mRNA were mixed and added to a 4-mm cuvette containing 0.4 mL of Caco-2-N cells (8×106) in Opti-MEM. Single electrical pulse was given with a GenePulser apparatus (Bio-Rad) with setting of 270V at 950μF. GFP signal can be observed 17 hours post electroporation. Three days post electroporation, P0 virus was collected and Caco-2-N cells were infected with P0 virus to amplify virus.
4
Transcription and Electroporation of JEV RNA
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The genome-length RNAs of parental JEV and Rluc-JEV were transcribed from the corresponding XhoI-linearized cDNA plasmids using T7 mMESSAGE mMACHINE Kit (Ambion). The transcription reactions were performed according to the manufacturer's protocols. For transfection, approximately 5 μg RNA was electroporated into 8 × 106 BHK-21 cells in 0.8 ml of ice cold PBS buffer (pH 7.5) in a 0.4 cm cuvette with the GenePulser apparatus (Bio-Rad) at 0.85 kV and 25 μF, pulsing three times at 3s intervals. After a 10-min recovery at room temperature, the transfected cells were mixed with 25 ml pre-warmed DMEM supplemented with 10% FBS, and were transferred into a T-75 flask and incubated at 37 °C with 5% CO2.
Virus titer and plaque morphology were determined by plaque assay. Briefly, a series of 1:10 dilutions were prepared, and 1 ml of viruses for each dilution were seeded onto each well of 6-well plates containing confluent BHK-21 cells (5× 105 cells/well, plated 1 day in advance). The plates were incubated at 37 °C with 5% CO2 for 1 h before the first layer of agar was added. After 3 days of incubation at 37 °C with 5% CO2, a second layer of agar containing neutral red was added. Plaques were photographed and counted after incubation of the plates for another 12 to 24 h.
Virus titer and plaque morphology were determined by plaque assay. Briefly, a series of 1:10 dilutions were prepared, and 1 ml of viruses for each dilution were seeded onto each well of 6-well plates containing confluent BHK-21 cells (5× 105 cells/well, plated 1 day in advance). The plates were incubated at 37 °C with 5% CO2 for 1 h before the first layer of agar was added. After 3 days of incubation at 37 °C with 5% CO2, a second layer of agar containing neutral red was added. Plaques were photographed and counted after incubation of the plates for another 12 to 24 h.
5
Protocols for Bacterial Transformation
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All strains and plasmids used in this study are listed in Table 1 . E. coli DH5α, TOP10 and TOP10F′ strains were grown in Luria Broth (LB) medium at 37°C with aeration. L. lactis strains were grown in M17 with 0.5% glucose (w/v) (GM17 media) at 30°C without aeration. Where appropriate, the media contained erythromycin (150 µg/ml for E. coli and 10 µg/ml for L. lactis), spectinomycin (50 µg/ml for E. coli and 300 µg/ml for L. lactis), or chloramphenicol (25 µg/ml for E. coli and 10 µg/ml L. lactis).
Electroporation of both E. coli and L. lactis was performed with a Gene Pulser apparatus (BioRad). E. coli transformants were recovered in SOC media (0.5% yeast extract, 2% tryptone, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCI2, 10 mM MgS04 and 20 mM glucose) for 1 h at 37°C with aeration. L. lactis transformants were recovered in GM17 medium with 0.5 M sucrose for 3 h at 30°C without aeration.
Electroporation of both E. coli and L. lactis was performed with a Gene Pulser apparatus (BioRad). E. coli transformants were recovered in SOC media (0.5% yeast extract, 2% tryptone, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCI2, 10 mM MgS04 and 20 mM glucose) for 1 h at 37°C with aeration. L. lactis transformants were recovered in GM17 medium with 0.5 M sucrose for 3 h at 30°C without aeration.
6
Cloning and Characterization of tet(L) Gene from S. anginosus
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The general procedures used for DNA manipulation were essentially those described by Sambrook and Russell (2001) . Restriction endonucleases (Fermentas, St. Leon-Rot, Germany), T4 DNA ligase (Roche, Mannheim, Germany), and Pfx DNA polymerase (Invitrogen, Carlsbad, CA, United States) were used according to the manufacturers’ instructions. The tet(L) gene from S. anginosus VA01-10AN was amplified by PCR with primers incorporating SalI and EcoRI restriction enzyme sites (Supplementary Table 1 ). Amplicons and pUC19 were digested with the two enzymes, ligated and electroporated into electrocompetent Escherichia coli DH10B cells using a GenePulser apparatus (Bio-Rad Laboratories, Richmond, CA, United States). Plasmid DNA from E. coli was purified as described by Sambrook and Russell (2001) , analyzed by restriction enzyme analysis and sequenced.
7
Construction of FADD and N-FADD Plasmids
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N-FADD (m-FADD, 1–181 aa), which is truncated of the C-terminal tail of mouse FADD, was amplified by primers N-FADD-F and N-FADD-R with pcDNA-FADD as template. Primers sequences as follows:
N-FADD-F: 5′-CGGGATCC ATGGACCCATTCCTGGTGC-3′
N-FADD-R: 5′-CCCAAGCTT TCACTGGGCTTCTTCCACCAG-3′
The sequence was cloned into eukaryotic expression vector pcDNA3.1 (−) after digested with BamHI and HindIII. The positive clone contains sequence of N-FADD named pcDNA-N-FADD. For hypoxia-inducible expression in VNP, the sequences of FADD and N-FADD were inserted into the prokaryote expression vector pQE30-NirB with BamHI, HindIII digestion and the resultant vectors were named pQE-NirB-FADD and pQE-NirB-N-FADD, respectively. All of the coding sequences of positive clones were confirmed to be correct by DNA sequencing (GenScript Corporation, Nanjing, Jiangsu, China). pQE-NirB, pQE-NirB-FADD and pQE-NirB-N-FADD plasmids were transformed into VNP using a Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) with conditions as follows: 2.5 kV, 25 μF and 400 Ω, respectively and plated on LB agar containing 100 μg/ml ampicillin. Positive VNPs were each named VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD.
N-FADD-F: 5′-CG
N-FADD-R: 5′-CCC
The sequence was cloned into eukaryotic expression vector pcDNA3.1 (−) after digested with BamHI and HindIII. The positive clone contains sequence of N-FADD named pcDNA-N-FADD. For hypoxia-inducible expression in VNP, the sequences of FADD and N-FADD were inserted into the prokaryote expression vector pQE30-NirB with BamHI, HindIII digestion and the resultant vectors were named pQE-NirB-FADD and pQE-NirB-N-FADD, respectively. All of the coding sequences of positive clones were confirmed to be correct by DNA sequencing (GenScript Corporation, Nanjing, Jiangsu, China). pQE-NirB, pQE-NirB-FADD and pQE-NirB-N-FADD plasmids were transformed into VNP using a Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) with conditions as follows: 2.5 kV, 25 μF and 400 Ω, respectively and plated on LB agar containing 100 μg/ml ampicillin. Positive VNPs were each named VNP-pN, VNP-pN-FADD and VNP-pN-N-FADD.
8
Cardiac Differentiation of Mouse ESCs
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Mouse AB1 ES cells were grown on mouse embryonic fibroblast (MEFs) feeders in standard ES culture medium. Briefly, ESCs were cultured on a feeder layer of Mitomycin C treated MEFs on 0.2% gelatin-coated cell culture dishes. Culture medium consisted of Dulbecco’s modified Eagle’s medium, 15% ES-grade fetal calf serum (FCS, Gibco), 1 mM sodium pyruvate (Gibco), 0.1 mM non-essential amino acids (Gibco), 0.1 mM β-mercaptoethanol (Sigma), 100 U/ml penicillin and 0.1 mg/ml streptomycin and 1000 U/ml leukemia inhibitory factor (LIF). Cardiac differentiation of ESCs was induced according to established procedures4 (link). Mouse embryonic fibroblasts (MEFs) and human keratinocyte cell lines were cultured in DMEM containing 10% FCS. Ago1−/−, 3−/−, 4−/− ES cells were kindly provided by X. Wang (Department of Biochemistry, Northwestern University, Evanston, USA). Electroporation was performed 1–2 days after plating (5 × 106 cells per plate) with 5 μg RNA using the Bio-Rad Gene Pulser apparatus (400 V, 250 F).
9
Identifying Clonal Correlation of poxtA-positive S. haemolyticus
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Multilocus sequence typing (MLST) was conducted for identification of clonal correlation of the poxtA-positive S. haemolyticus (http://www.shaemolyticus.mlst.net Accessed on: 29 January 2021) [26 (link)]. Plasmid DNA from all poxtA-positive CoNS isolates was extracted using a Qiagen Prep Plasmid Midi Kit (Qiagen, Hilden, Germany) and transferred into a recipient S. aureus strain RN4220 by electroporation using Gene Pulser apparatus (Bio-Rad, Hercules, CA, United States) [27 (link)]. Electrotransformants were selected on brain heart infusion (BHI) agar containing 10 µg/mL of florfenicol. Electrotransformants were further confirmed for the presence of poxtA gene by PCR analysis. The successful electrotransformants were further screened for the presence of aadD, fexB, tet(L) and tet(M) genes by PCR.
10
Metabolic Engineering of C. glutamicum
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Chromosomal DNA was isolated from Z. mobilis ZM4 and used as PCR template to amplify eda, zwf, and edd. Primers used in the present study are listed in Additional file 2 : Table S1. PCR was performed using a GeneAmp PCR 9700 system (Applied Biosystems, USA) and PrimeStar HS DNA polymerase (Takara, Japan). The tac promoter was fused to the 5′ end of each gene and the resulting DNA fragments were integrated into C. glutamicum R strain-specific islands in the chromosome, as described previously [30 (link)]. Markerless pfkA disruption was performed according to a method described previously, which is based on homologous recombination, followed by sacB selection [21 (link)]. Gene disruption was confirmed by PCR. Transformation of C. glutamicum was performed by electroporation by delivering an electrical pulse of 2.5 kV, 200 Ω resistance, and 25 µF capacitance in a 0.1-cm cuvette using a Gene Pulser apparatus (Bio-Rad, USA).
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