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13 protocols using dna gel extraction kit

1

Characterization of crdR Binding to crd Promoters

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DNA fragments containing various lengths of the crd promoter (crdP, crdP142, crdP108, crdP98, crdP53, crdP13, and crdP1) and ~450 bp upstream of the start codon (ATG) of crdR (relA(AGRO_1479) celA (AGRO_4469), and crdS (AGRO_1848) named relAP celAP and crdSP were obtained by PCR amplification with primers listed in Table 2 respectively, those fragments were purified with a DNA gel extraction kit (Sangon Biotech) respectively according to the manufacturer’s protocol. A electrophoretic mobility shift (EMSA) binding assay was performed as previously described with slight modifications [40 (link)]. Briefly, 10 μL of 0.25–0.50 mg/mL purified His-tagged crdR in 4× EMSA buffer (15 mM HEPES, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 1 μg/mL poly dI-dC) was incubated with 10 μL of different purified target DNA fragments (0.5 μM) in ddH2O at room temperature for 30 min. DNA-protein complexes were loaded onto a 2% agarose gel and separated at 80 V for 1.5 h, and the gel was stained with SYBR Green I and visualized with a UV trans-illuminator (Upland, CA).
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2

qRT-PCR Analysis of P. rockii Genes

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The qRT-PCR primers were designed by Oligo6.0, and P. rockii’s 18S-26S internal transcribed spacer (18S-26S ITS) gene [57 (link)] was used as the internal reference gene, as shown in Table S1. RNA from the seeds and other organs was extracted using the RNAprep Pure Plant kit (Tiangen Beijing, China); subsequently, approximately 1 µg of total RNA was used for reverse transcription, using the PrimeScriptTM RT reagent Kit (TaKaRa Dalian, China). Three copies of each sample were analyzed. The relative expression analyses were performed by the 2−ΔΔCT values [58 (link)]. The reverse transcription of the P. rockii cDNA was used as a template for PCR amplification. The PCR products were purified by the DNA Gel Extraction Kit (Sangon), and then, the amplified PCR fragments were inserted into the pMD19-T vector. After that, positive clones were screened for gene sequencing. The correctly sequenced plasmids were stored in the refrigerator at −20 °C for later usage.
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3

Optimized CRISPR-dCas9-VPR System

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DNA primers were ordered from Sangon Biotechnology (Shanghai, China). Escherichiacoli strain DH5α was obtained from MiaolingBio (Wuhan, China). SpCas9 nuclease, Phanta Max Super-Fidelity DNA Polymerase, Hiscript III RT SuperMix for quantitative polymerase chain reaction (qPCR), T7 High Yield Transcription Kit, iTaq Universal SYBR Green Supermix and miRNA 1st Strand cDNA synthesis kits were purchased from Vazyme (Nanjing, China). Lipofectamine 3000 was purchased from ThermoFisher (USA). Plasmid DNA was extracted using the Plasmid mini-prep kit (Vazyme), and PCR amplicons were purified using the DNA gel extraction kit or the PCR product purification kit (Sangon Biotechnology). T4 ligase and restriction enzymes were purchased from New England Biolabs. Vectors of pJ23119, pU6, pCMV and pACYC plasmids were obtained from Addgene. The HEK293T-dCas9-VPR was prepared through lentivirus infections as described in our previous work (26 (link)). Trizol reagent was purchased from Ambion (USA). The firefly Luciferase Reporter Gene Assay Kit was purchased from Beyotime (China). The Annexin V-FITC/PI apoptosis detection kit was purchased from BestBio (China). Venetoclax was purchased from MedChemExpress (MCE). Other reagents were obtained from Sangon Biotechnology, unless otherwise indicated.
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4

PCR Amplification and Sequencing Protocol

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PCR amplification products were purified using the PCR purification kit (Takara Japan). PCR amplification was performed for 30 cycles of pre-denaturation for 3 min at 98°C, followed by 30 sec at 98°C (denaturation), 45 sec at 55°C (annealing) and 1 min at 72°C (extension), and a final extension at 72°C for 10 min.
PCR products were purified using a PCR product purification kit (Shanghai Shengong, China). PCR products were electrophoretically size fractionated on 1.5% agarose gels that contained ethidium bromide and were visualized with UV light. The DNA fragment was gel-extracted using the DNA Gel Extraction Kit (Sangon Biotech). Purified PCR products were further used in the sequencing reaction process. The program used was an initial denaturation step at 96°C for 1 min, followed by 25 cycles (10 s of denaturation at 96°C, 50 s of annealing at 55°C, and 4 min of extension at 60°C) and a final extension at 60°C for 4 min. Cycle sequencing reactions were then purified using NaAc/EDTA precipitation. The sample was purified by ethanol and then dissolved in deionized formamide to undergo sequencing.
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5

Cloning and Sequencing of Lignocellulose-Degrading Enzymes

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Using cDNA obtained by reverse transcription as a template, RT-PCR amplification of xynF1 (NCBI ID: 4,980,082), abnC (NCBI ID: 4,979,546), cbhC (NCBI ID: 4,982,491), bglM (NCBI ID: 4,984,238), and eglD (NCBI ID: 4,988,091) was carried out using gene-specific primers. The PCR products were purified using the DNA Gel Extraction Kit (Sangon Biotech Co., Ltd., Shanghai, China), ligated into the One Step ZTOPO-Blunt/TA vector (Zhuangmeng Technology Co., Ltd., Beijing, China) and transformed into Escherichia coli DH5α. The clones were verified by DNA sequencing (Sangon Biotech Co., Ltd., Shanghai, China). Then, intron-spanning primers were designed for PCR validation based on the five lignocellulose-degrading enzyme genes xynF1, abnC, cbhC, bglM and eglD. The primers are listed in the Supplemental Table S1.
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6

Generating T7 RNAP Insertion Mutant

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The λ Red homologous recombination system was simplified to generate a lon deletion and T7 RNAP insertion mutation of SE. In brief, primers P1 and P2 containing 50-nt homology arms targeting the lon gene and the genome of BL21(DE3) as a PCR template were used to amplify a 3306-bp linear PCR product. The resulting PCR product was purified by DNA Gel Extraction Kit (Sangon Biotech, Guangzhou, China). SE competent cells were prepared by us, and the pKD46 plasmid was transformed into SE competent cells by electroporation under the conditions of 200 Ω, 25 µF, and 2000 V. An SE recombination strain containing the pKD46 plasmid was selected and put into LB liquid medium with 100 µg/mL Amp+ at 30 °C. When the OD600 value reached 0.2–0.3, L-arabinose (L-ara) with a final concentration of 20 mM was added to bacterial cells until the OD600 value reached 0.6. Then, the cells were prepared into electrocompetent cells followed by transformation with approximately 100 ng of the purified PCR product. An SE recombinant inbred strain with T7 RNAP gene insertion and a lon gene deletion named SE/Δlon::T7 RNAP/pKD46 were identified by PCR using primers P3 and P4 according to the nucleotide sequence of SE available in NCBI (NZ_CP012347.1). The positive recombinant strain was cultured in LB broth at 42 °C for 16 h to remove the pKD46 plasmid.
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7

Bacterial 16S rRNA Gene Sequencing

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DNA was extracted from the inner part of the fecal samples (0.5 g) by using the EZNA Soil DNA Kit (D5625–01; OmegaBio-Tek, Inc., Norcross, USA) according to manufacturer’s instructions. Subsequently, DNA was amplified using the V3–V4 hypervariable regions of the bacterial 16S rRNA gene barcoded (unique 7nt) primers fusion 341 F primer: CCTACACGACGCTCTTCCGATCTN(barcode)CCTACGGGNGGCWGCAG and fusion 805 R primer: GACTGGAGTTCCTTGGCACCCGAGAATTCCAGACTACHVGGGTATCTAATCC). The polymerase chain reaction (PCR) reaction mixture (50 μL) contained 5 μL 10× buffer, 0.5 μL dNTPs (10 mM each), 10 ng genomic DNA, 0.5 μL Bar-PCR primer F (50 μM), 0.5 μL Primer R (50 μM), 0.5 μL Plantium Taq (5 U/μL), and 43 μL molecular biology grade water. PCR cycles included 94 °C for 3 min; 5 cycles of 94 °C for 30 s, 45 °C for 20 s, and 65 °C for 30 s; 20 cycles of 94 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s; and a final extension at 72 °C for 5 min. The amplicons were subsequently purified using a DNA gel extraction kit (SK8131, Sangon Biotech Co. Ltd., Shanghai, China), and the purified amplicons were paired-end (PE) sequenced (2 × 300) by using the Illumina MiSeq platform at Sangon Biotech Co. Ltd (Shanghai, China).
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8

Bacterial 16S rRNA Gene Amplification

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About 1.5 g of the surface-sterilized plant sample was frozen with liquid nitrogen and ground to a fine powder in a sterilized and precooled mortar. DNA was extracted using a Plant DNA Kit (Omega Bio-Tek, Norcross, USA) and the V3-V4 hypervariable region of the bacterial 16S rRNA gene amplified using Bac 341f (5′-CCTACACGACGCTCTTCCGATCTN-3′) and Univ 805r (5′-GACTGGAGT TCCTTGGCACCCGAGAATTCCA-3′) primers. The 50 μl PCR reaction mixture contained 100 ng of DNA extract, 1× Taq reaction buffer, 20 pmol of each primer, 200 μM each dNTP and 1.5 U of Taq DNA polymerase (Sangong Biotech, China). DNA aliquots were PCR-amplified with 5 min denaturation at 95 °C, 30 cycles of 1 min at 94 °C, 50 s at 55 °C, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. All samples were amplified. Replicate PCR products of the same sample were assembled within a PCR tube. Then they were visualized on agarose gels (2% in TBE buffer) containing ethidium bromide, and purified with a DNA gel extraction kit (Sangong Biotech, China). After purification, the concentration of the PCR products was measured on Qubit®2.0 Fluorometer using the PicoGreen® dsDNA quantitation assay (Invitrogen, Carlsbad, CA). DNA concentration was adjusted to 1 ng/ml. The amplicon libraries were prepared by pooling 10 ng of each PCR.
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9

Yeast Genomic DNA Extraction and Manipulation

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DNA restriction enzymes (BamHI, NotI, and SacI) were purchased from Takara Biomedical Technology Co., Ltd (Beijing, China). The Rapid Yeast Genomic DNA Isolation Kit, Plasmid DNA Mini-preps Kit, SDS-PAGE Preparation Kit, DNA Markers, Protein Markers, DNA Gel Extraction Kit, and the primers for α-factor, 5′-AOX, and 3′-AOX were supplied by Sangon Biotech Co., Ltd (Shanghai, China). TS GelRed Nucleic Acid Dye and T5 Super PCR Mix were obtained from Beijing Tsingke Biotech Co., Ltd (Beijing, China). Hemin (CAS: 16009-13-5, purity: 98%) and biliverdin hydrochloride (CAS: 55482-27-4, purity: 97%) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). Geneticin (G418) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). All other reagents were commercially available in analytical and biological grades.
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10

EMSA Analysis of QseB Binding

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The DNA fragments used for EMSA were obtained by PCR using p-primers from the chromosome of the WT. The p-primers were synthesized by the manufacturer (TsingKE). The p-primers used in EMSA are listed in Table 2. Binding reactions were performed with a total of 150 fmol each probe mixed with various amounts of purified QseB in 4 µl 5 × binding buffer (Beyotime, Shanghai, China) at room temperature for 30 min. Afterward, 5 µl gel loading buffer (0.25 × TBE, 60%; glycerol, 40%; and bromophenol, 0.2% (wt/vol)) was added, and mixtures were electrophoresed in a 6% native polyacrylamide gel in 0.5 × TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0). The result of promoter fragments was purified with a DNA Gel Extraction Kit (Sangon, Shanghai, China), and a chemiluminescent EMSA kit (Beyotime, Shanghai, China) was used to detect the signals of DNA-protein complexes according to the manufacturer’s instructions.
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