A high titer stock of ZCSE2 (≈1010 PFU/mL) was prepared as described above. To obtain ZCSE2 genomic DNA, phages were initially treated with proteinase K (100 μg/mL in 10 mM EDTA (pH 8)). DNA was extracted from the preparation using DNA Wizard Kit (Promega, United Kingdom) according to the manufacturer’s instructions. Library preparation of ZCSE2 genomic DNA followed the Illumina Nextera tagmentation protocol (Illumina, Cambridge, United Kingdom) and the library sequenced using the Illumina v3 sequence cassette for 600 cycles on the MiSeq platform. The data were composed of 3.1 million paired-end sequence reads with lengths of 250 bp. De novo assembly of sequence reads was performed using CLC Genomics Workbench version 11.0.1 (Qiagen, Aarhus, Denmark). Assembled reads yielded a complete double-stranded DNA ZCSE2 genome of 53,965 bp (coverage around 7000-fold). Gene predictions were made using PHASTER [27 (link)] and HHpred [28 (link)] to identify putative open reading frames (ORFs), followed by manual curation and polishing with Artemis and BLAST (non-reductive NCBI databases). The sequence is available under the GenBank accession number MK673511. Bacteriophage genome sequence alignments, average nucleotide identities, and phylogenetic relationships were calculated using CLC Genomics Workbench 20.0.3.
Wizard dna kit
Manufactured by Promega
Sourced in United States, United Kingdom
The Wizard DNA kit is a laboratory product designed for the extraction and purification of DNA. It provides a reliable and efficient method for isolating DNA from various sample types. The kit includes necessary reagents and materials to facilitate the DNA extraction process. Its core function is to enable the isolation of high-quality DNA samples for further analysis and experimentation.
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Lab products found in correlation
Clc genomics workbench version 11, by Qiagen (2 mentions)
V3 sequence cassette, by Illumina (1 mentions)
Nextera tagmentation protocol, by Illumina (1 mentions)
Plasmid miniprep kit, by Qiagen (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Clc genomics workbench version 10, by Qiagen (1 mentions)
Bio plex, by Bio-Rad (1 mentions)
Truseq nano kit, by Illumina (1 mentions)
Simpliamp, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Seqman ngen, by DNASTAR (1 mentions)
Strataclone pcr cloning kit, by Agilent Technologies (1 mentions)
Miseq platform, by Illumina (1 mentions)
Massarray platform, by Labcorp (1 mentions)
13 protocols using wizard dna kit
1
Sequencing and Assembly of ZCSE2 Phage Genome
2
Evaluating P. gingivalis Virulence Factors
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Balb/c mice were dorsally injected with equal numbers (1.5 × 109) of P. gingivalis 33277 and of the respective mutant strain. Mice were monitored daily and abscesses were collected 4–5 days post-infection. DNA was isolated using a DNA wizard kit (Promega), and amplified by qPCR with primers to 33277 16S rRNA or the appropriate antibiotic resistance gene (Supplementary Table 1 ). Numbers of P. gingivalis were calculated by comparison with a standard curve derived from known amounts of P. gingivalis or the respective mutant using 16S rRNA and antibiotic resistant primers. Competitive index (CI) was calculated as the output ratio of mutant to parent divided by the input ratio of mutant to parent, and significance determined by the Wilcoxon signed rank test.
3
Bacterial Strain Construction and Transformation
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Escherichia coli DH5α was used for plasmids amplification. Bacillus subtilis strains were mutant derivatives of 168CA and their relevant characteristics are shown in Table 1. All the chemicals and reagents used in this work were obtained from Sigma except where stated otherwise.
Plasmid DNA was routinely extracted using Plasmid MiniPrep Kit (Qiagen), whilst the extraction of B. subtilis chromosomal DNA was either done using DNA wizard kit (Promega) for sequencing and PCR, or as a simple DNA extract (47) to permit strain construction. The transformation of B. subtilis strains was carried out according to the method developed by Anagnostopoulos and Spizizen (48) , modified by Young and Spizizen ( 49) and E. coli transformation was done as described by Hanahan,1985 (50) . Selection for antibiotic resistance markers was done using the following concentrations: chloramphenicol (5 µg/ml), erythromycin (1 µg/ml), spectinomycin and ampicillin (100 µg/ml) in Nutrient Agar (Oxoid). Where required 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was added to a final concentration of 0.002% to provide a visual indication of β-galactosidase activity, and where required IPTG was added to a final concentration of 1 mM.
Plasmid DNA was routinely extracted using Plasmid MiniPrep Kit (Qiagen), whilst the extraction of B. subtilis chromosomal DNA was either done using DNA wizard kit (Promega) for sequencing and PCR, or as a simple DNA extract (47) to permit strain construction. The transformation of B. subtilis strains was carried out according to the method developed by Anagnostopoulos and Spizizen (48) , modified by Young and Spizizen ( 49) and E. coli transformation was done as described by Hanahan,1985 (50) . Selection for antibiotic resistance markers was done using the following concentrations: chloramphenicol (5 µg/ml), erythromycin (1 µg/ml), spectinomycin and ampicillin (100 µg/ml) in Nutrient Agar (Oxoid). Where required 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) was added to a final concentration of 0.002% to provide a visual indication of β-galactosidase activity, and where required IPTG was added to a final concentration of 1 mM.
4
Characterization of Bacteriophage ZCEC5
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Bacteriophage ZCEC5 was examined using transmission electron microscopy at the National Research Center (Cairo, Egypt) as previously described (Atterbury et al. 2003 (link)). Briefly, fixed phages on Pioloform grids using glutaraldehyde were negatively stained with 0.5% uranyl acetate. After drying, the specimens were examined using a JEOL 100CX transmission electron microscope.
Genomic DNA was extracted from a lysate of phage ZCEC5 (1010 PFU mL−1) treated with proteinase K (100 μg mL−1 in 10 mM EDTA at pH 8) before purification by the Wizard DNA kit (Promega, UK) according to the manufacturer’s instructions. The genome DNA of phage ZCEC5 was sequenced from libraries prepared using the Illumina tagmentation protocol on the MiSeq platform. The data was composed of 0.52 million paired-end sequence reads with read lengths of approximately 250 bp. The data was de novo assembled using CLC Genomics Workbench version 10.0.1 (Qiagen, Aarhus, Denmark). The open reading frames (ORFs) were predicted from PHASTER (Arndt et al. 2016 (link)). The genome DNA sequence appears in GenBank under the Accession Number MK542015.
Genomic DNA was extracted from a lysate of phage ZCEC5 (1010 PFU mL−1) treated with proteinase K (100 μg mL−1 in 10 mM EDTA at pH 8) before purification by the Wizard DNA kit (Promega, UK) according to the manufacturer’s instructions. The genome DNA of phage ZCEC5 was sequenced from libraries prepared using the Illumina tagmentation protocol on the MiSeq platform. The data was composed of 0.52 million paired-end sequence reads with read lengths of approximately 250 bp. The data was de novo assembled using CLC Genomics Workbench version 10.0.1 (Qiagen, Aarhus, Denmark). The open reading frames (ORFs) were predicted from PHASTER (Arndt et al. 2016 (link)). The genome DNA sequence appears in GenBank under the Accession Number MK542015.
5
Genetic Profiling of Inflammatory Cytokines
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Peripheral blood mononuclear cells (PBMC) and sera were collected, separated and stored at −80 °C under endotoxin-minimised conditions. DNA was extracted from PBMC (Wizard DNA kit; Promega) and quantified using Nano-DropR ND-1000 (Biolab), with quality verified by agarose gel electrophoresis. Genomic DNA was sent to the Australian Genome Research Facility (AGRF) for genotyping (Sequenom MassARRAY) of functional SNPs in pro- and anti-inflammatory cytokine genes implicated in the acute sickness response, including IFN-γ (+874 T/A, rs2430561), TNF-α (−308 G/A, rs1800629), IL-1β (−511 C/T, rs16944), IL-6 (−174 G/C, rs18000795) and IL-10 (−1082 G/A, rs1800896 and -592 C/A, rs1800872).
Sera from the end-treatment time point were subsequently thawed and concentrations of the cytokines, IL-1β, IL-6, IL-10, TNF-α, and IFN-γ analysed in a bead-based multiplex immunoassay system (BioPlex; BioRad, Hercules, CA).
Sera from the end-treatment time point were subsequently thawed and concentrations of the cytokines, IL-1β, IL-6, IL-10, TNF-α, and IFN-γ analysed in a bead-based multiplex immunoassay system (BioPlex; BioRad, Hercules, CA).
6
Phage Genome Sequencing Protocol
7
Antibiotic Resistance Gene Detection
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For detection of known antibiotic resistance genes, primers and PCR protocols previously described for blaCTX-M, [28 (link)], blaSHV and blaTEM [29 (link)], blaNDM [30 (link),31 (link)],blaOXA [32 (link)] and blaVIM [33 (link)] were used (Table S1 ). Bacterial DNA was extracted using the Wizard DNA kit (Promega, Madison, WI, USA) and PCR amplifications were done in a SimpliAmp thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA).
8
Comparative Genomics of L. johnsonii Strains
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L. johnsonii strains LB1 and NCK88, a well-studied L. johnsonii isolate, were each inoculated from frozen glycerol stocks into sterile MRS and incubated anaerobically at 37°C overnight. Cells were precipitated by centrifugation and genomic DNA was extracted using Promega’s Wizard DNA kit. Whole genome sequencing was performed by the Penn State Genomics Core Facility using an Illumina MiSeq instrument with 300 bp paired end reads. Sequencing data was assembled de novo with DNASTAR’s SeqMan NGen (DNASTAR Inc.), and contigs were mapped against the reference L. johnsonii NCC533 genome with progressiveMauve [44 (link),45 (link)]. Genomes were compared with WebACT and dotplots of translated open reading frames were generated with PROmer in Galaxy [46 (link)–50 (link)]. Sequence data is available under BioProject ID PRJNA315676.
9
Phage Lysate Preparation and Genome Sequencing
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High-titer phage lysates were prepared by diluting a lysate 10-fold into phage buffer with 0.1 M CaCl2 and plating 10 µl of the 10−2 dilution with 250 µl of mc2155 and 3 ml of liquid Middlebrook 7H9 medium. All plates were incubated at 37°C for 18 to 24 h. Webbed plates were flooded with 4 ml of phage buffer and incubated at room temperature for at least 4 h. Lysates were collected, filtered through a 0.22 μm-pore-size filter, and then stored at 4°C. The phage genome sequences have been reported previously or are available in GenBank. A Phamerator database (Actinobacteriophage_Draft, August 2018) was used for comparative genomic analyses (35 (link)). Phage genomic DNA was extracted using either a Wizard DNA kit (Promega) or phenol-chloroform as described previously (36 (link)) and was completely sequenced using Illumina MiSeq 150-base single-end runs and assembled as described previously (11 (link)).
10
ESBL Gene Amplification and Sequencing
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Amplification of ESBL genes was performed using previously published primers and protocols for blaCTX [30 (link)], blaSHV [31 (link)], blaTEM [32 ], and blaNDM genes [33 (link),34 (link)]. Bacterial DNA was extracted using Wizard DNA kit (Promega, Madison, WI, USA) was used as template for PCR amplification in a Hybaid thermal cycler (Thermo Fisher Scientific Inc., Waltham, MA, USA). For confirmation, representative PCR products were cloned using Strataclone PCR cloning kit (Agilent, Santa Clara, CA, USA) and sequenced (Xcelris Labs, Ahmedabad, India).
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