The porcine IFNs and anti-viral genes were searched from NCBI data base and designed using GENETYX SV/RC ver 13 (GENETYX, Tokyo, Japan), considering appropriate Tm-values and GC% for forward and reverse primer (Supplementary Table S1 ). The synthesized primers were used to amplify the gene of interest in a PCR-based Ex Taq (Takara Bio Inc., Shiga, Japan). Amplified DNA fragments were purified by using NucleoSpin Gel and PCR Clean-up (Takara Bio) and then inserted into pGEM T-Easy Vector (Promega, Madison, WI, USA) to obtain plasmids. Plasmids were mixed with 10 × KCM (K, Ca, Mg), 30% PEG (polyethylene glycol) and sterile water. The solution was used to treat E. coli JM109 competent cells (Takara Bio) on ice for 30 min. E. coli cells were incubated on TSB agar medium supplemented with 100 μg/mL (γ) Amp, X-gal (Takara Bio), and IPTG (Takara Bio) at 37 °C for 18 h. White colonies were selected and the presence or absence of insertions of the genes of interest into the vectors were confirmed by colony-PCR. Positive colonies were cultured in TSB medium supplemented with /100γ Amp at 37 °C for 18 h, and then the plasmids were extracted using Fast-Gene Plasmid Mini Kit (Nippon Genetics, Tokyo, Japan).
X gal
Manufactured by Takara Bio
Sourced in Japan
X-gal is a colorimetric substrate used in molecular biology and microbiology for the detection and screening of beta-galactosidase activity. It is a synthetic organic compound that produces a blue colored product when cleaved by the beta-galactosidase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
E coli jm109 competent cells, by Takara Bio (1 mentions)
Nucleospin gel and pcr clean up, by Takara Bio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Takara Bio (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Fastgene plasmid mini kit, by Nippon Genetics (1 mentions)
Chloramphenicol, by Fujifilm (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Bacto tryptone, by Thermo Fisher Scientific (1 mentions)
Bacto yeast extract, by BD (1 mentions)
L arabinose, by Fujifilm (1 mentions)
Difco m9 minimal salts, by BD (1 mentions)
Bacto agar, by Fujifilm (1 mentions)
D maltose monohydrate, by Fujifilm (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Bacto agar, by BD (1 mentions)
Dntp mixture, by Takara Bio (1 mentions)
Dna gel extraction kit, by Takara Bio (1 mentions)
Ex taq polymerase, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
10 protocols using x gal
1
Cloning and Transformation of Porcine IFN and Anti-Viral Genes
2
Genomic Analysis of E. coli Strains
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The E. coli strains and their mutants used in this study are listed in Table 1 . Whole-genome sequences of the 442 O121:H19 strains (Data set S1) previously used in our phylogenetic analysis (13 ) were used for the analysis of IS insertion into the lacZ and iee genes.
Bacteria were grown in the following media: LB (1% [wt/vol] Bacto Tryptone, Gibco; 0.5% [wt/vol] Bacto Yeast Extract, Becton, Dickinson [BD]; 1% [wt/vol] sodium chloride, nacalai tesque), LB agar (LB containing 1.5% [wt/vol] Bacto Agar, BD), MAC (Difco MacConkey agar base, BD; 1% [wt/vol] lactose monohydrate, Wako), MAC not supplemented with lactose (Difco MacConkey agar base), Pearlcore MAC (Pearlcore MacConkey agar, Eiken Chemical Co.), MM (Difco M9 Minimal Salts, BD; 2 mM magnesium sulfate heptahydrate, Wako; 0.1% D-[+]-glucose, nacalai tesque), and MM agar (MM containing 1.5% [wt/vol] Bacto Agar). The growth media were supplemented with regents and antibiotics when necessary at the following concentrations: L(+)-arabinose (Wako), 1 mM; IPTG (Wako), 0.3 mM or 30 mM; X-gal (TaKaRa), 40 μg/mL; sucrose (nacalai tesque), 10% (wt/vol); D-(+)-glucose, 0.1%, 0.2%, or 0.4% (wt/vol); lactose monohydrate, 1.0% (wt/vol); D(+)-maltose monohydrate (Wako), 1.0% (wt/vol); chloramphenicol (Wako), 20 μg/mL; ampicillin (Sigma), 50 μg/mL; tetracycline (nacalai tesque), 10 μg/mL.
Bacteria were grown in the following media: LB (1% [wt/vol] Bacto Tryptone, Gibco; 0.5% [wt/vol] Bacto Yeast Extract, Becton, Dickinson [BD]; 1% [wt/vol] sodium chloride, nacalai tesque), LB agar (LB containing 1.5% [wt/vol] Bacto Agar, BD), MAC (Difco MacConkey agar base, BD; 1% [wt/vol] lactose monohydrate, Wako), MAC not supplemented with lactose (Difco MacConkey agar base), Pearlcore MAC (Pearlcore MacConkey agar, Eiken Chemical Co.), MM (Difco M9 Minimal Salts, BD; 2 mM magnesium sulfate heptahydrate, Wako; 0.1% D-[+]-glucose, nacalai tesque), and MM agar (MM containing 1.5% [wt/vol] Bacto Agar). The growth media were supplemented with regents and antibiotics when necessary at the following concentrations: L(+)-arabinose (Wako), 1 mM; IPTG (Wako), 0.3 mM or 30 mM; X-gal (TaKaRa), 40 μg/mL; sucrose (nacalai tesque), 10% (wt/vol); D-(+)-glucose, 0.1%, 0.2%, or 0.4% (wt/vol); lactose monohydrate, 1.0% (wt/vol); D(+)-maltose monohydrate (Wako), 1.0% (wt/vol); chloramphenicol (Wako), 20 μg/mL; ampicillin (Sigma), 50 μg/mL; tetracycline (nacalai tesque), 10 μg/mL.
3
X-gal Staining for Senescent Cells
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A total of 2 × 104 cells were seeded on 6-well plates and cultured overnight. The cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 5 minutes. After washing with PBS, the cells were stained with a solution containing 1 mg/ml X-gal (Takara), 40 mM citric acid/Na phosphate buffer, 5 mM K4[Fe(CN)6] 3 H2O, 5 mM K3[Fe(CN)6],150 mM sodium chloride and 2 mM magnesium chloride (pH 6.0) overnight. The cells were incubated at 37°C for 1 day and were photographed. At least 80 cells from more than 5 fields were counted.
4
Bacterial Dual-Plasmid Assay for STnc1480
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The interaction between STnc1480 and its target genes was analyzed using the bacterial dual-plasmid reporter system. In brief, F4/R4 and F5/R5 primers were used to amplify the operon DNA sequences of STnc1480 and lpfA. Then, the pUT18C-STnc1480 and pMR-LacZ-lpfA plasmids were constructed; the constructed recombinant plasmids were co-transfacted into BTH101 E. coli competent cells via electroporation. The positive clones on a Kan r (100 mg/ mL) Amp r (100 mg/ 9mL) (BIOTOPPED, China) double-resistant plate coated with X-gal (TaKaRa Bio, Inc., Japan) and Isopropyl-beta-Dthiogalactopyranoside(IPTG) (Solarbio Science & Technology Co., Ltd., China) were screened out and placed them at 37 o C overnight and the color change of the bacterial lawn was observed.
5
RNA Extraction and PCR Amplification
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The RNA PCR Kit (AMV) Ver. 3.0 reverse transcription kit was purchased from TaKaRa (Shiga, Japan); the viral RNA Kit was purchased from OMEGA Bio-Tek (Norcross, GA, USA); T4 DNA ligase, EX Taq polymerase, dNTP mixture, isopropyl b-d-1-thiogalactopyranoside, X-Gal, l-EcoT14I marker, DNA Gel Extraction Kit, and transfection kit (Lipofectamine TM 2000) were purchased from Takara Biotechnology Co., Ltd. (Dalian, China) The E.Z.N.A. ® HP Plasmid Midi Kit (OMEGA Bio-Tek) was used for large-scale plasmid extraction. Other agents were imported or domestic analytical reagents.
6
Detecting SsSte12-SsMcm1 Interaction
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The interaction between SsSte12 and SsMcm1 was detected with Matchmaker Gold Y2H system (Clontech, Japan) and BiFC system as described previously (Wang et al., 2017 (link)). For Y2H assay, both SsSte12 and SsMcm1 (SS1G_05588) coding regions were cloned into pGBKT7 and pGADT7 for reciprocal protein–protein interaction confirmation, respectively. The resulting constructs were co-transformed into Y2H strain Y187 with different combinations according to the manufacturer’s instructions. The transformed yeast cells were plated on SD-Leu-Trp and were identified by PCR. Subsequently, the positive transformants were grown on the SD-Leu-Trp-His-Ade medium supplemented with X-Gal according to the yeast protocol handbook (Clontech, Japan).
For the BiFC assay, the coding regions of SsSte12 and SsMcm1 were fused into cYFP (pSAT4-cEYFP-N1) and nYFP(pSAT4-nEYFP-N1) vectors, respectively. The resulting constructs were used for Arabidopsis protoplast transformation via PEG/Ca2+-mediated method as described previously (Yoo et al., 2007 (link)). The fluorescent signal and localization of SsSte12 and SsMcm1 fusion proteins were detected using a confocal laser scanning microscope (Nikon ECLIPSE Ts2R, Melville, NY, United States). Excitation wavelength was 514 nm and detection rage of emission wavelength was 520–550 nm.
For the BiFC assay, the coding regions of SsSte12 and SsMcm1 were fused into cYFP (pSAT4-cEYFP-N1) and nYFP(pSAT4-nEYFP-N1) vectors, respectively. The resulting constructs were used for Arabidopsis protoplast transformation via PEG/Ca2+-mediated method as described previously (Yoo et al., 2007 (link)). The fluorescent signal and localization of SsSte12 and SsMcm1 fusion proteins were detected using a confocal laser scanning microscope (Nikon ECLIPSE Ts2R, Melville, NY, United States). Excitation wavelength was 514 nm and detection rage of emission wavelength was 520–550 nm.
7
Yeast One-Hybrid System for Wx Promoter
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The Clontech™ one‐Hybrid System (Clontech, Dalian, China) was used in this study. The CDS of potential transactivators were fused with GAL4 AD domain in pB42AD (Clontech, Dalian, China), and the promoter region of Wx were cloned into pLacZ2u (Clontech, Dalian, China). Yeast strain EGY48 was transformed with indicated plasmids and grew on SD/‐Ura/‐Trp plates, and then strike on SD/‐Ura/‐Trp plates containing 2% glactose, 1% raffinose, 1 × BU salts and 80 mg/L X‐Gal (Clontech, Dalian, China). The interaction was confirmed by the visualization of blue colonies on the medium. NF‐YB1‐pB42AD and SUT4‐pLacZ2u were used as CK+ (Bai et al., 2015 ). The empty vectors pLacZ2u and pB42AD were used as negative control.
8
Yeast Two-Hybrid Assay for Protein Interactions
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All of the constructs in pGBKT7 and pGADT7 were used to transform AH109 yeast cells (Clontech) by using LiAc methods [29] (link) to test self-interactions and interactions between AtGRIPaa1-604 and AtGRIPaa605–788. Colonies were selected on SD (synthetic drop-out medium)/-Trp/-Leu medium, and the selected cells were then streaked on SD/-Trp/-Leu/-His/-Ade plates to test their interactions. Positive yeast transformants were tested with X-gal (Clontech) to detect MEL1 reporter gene expression.
9
Yeast-based DROT1 promoter analysis
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The DROT1 promoter amplified from IRAT109 and Yuefu was inserted into pLacZi2μ vector to generate proDROT1109:: LacZ and proDROT1YF:: LacZ reporter constructs, respectively. The full-length CDSs of ERF3 and ERF71 were amplified from the cDNA of Nipponbare and inserted into pB42AD to generate AD-ERF3 and AD-ERF71, respectively. The fused AD and LacZ plasmids were co-transformed into yeast strain EGY48. The transformants were grown on SD/-Trp-Ura dropout media containing 20 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) according to the manufacturer′s instructions (Clontech).
10
Yeast Two-Hybrid Screening for Protein Interactions
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The indicated vectors were co-transformed into yeast strain Y190, and positive clones were selected on synthetic defined (SD)/-Leu/-Trp medium. Randomly selected clones were cultured on SD/-Leu/-Trp/-His/-Ade medium and stained with 40 mg/ml X-Gal (Clontech) to determine b-galactosidase activity. Coding sequences (CDS) of genes were amplified and ligated into pGADT7 or pGBKT7. Plasmids carrying EDR4 and its truncated forms were described previously (Wu et al., 2015) .
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