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9 protocols using pureyield plasmid miniprep

1

Cloning and Sequencing of Bisulfite-Treated DNA

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The purified amplicons were cloned into the TOPO TA Cloning® vector (Invitrogen, CA, USA) and transferred into DH5α cells using a heat shock procedure. Plasmid DNA was isolated using Pure Yield Plasmid Miniprep (Promega, Madison, WI, USA), and individual clones were sequenced using BigDye® cycle sequencing chemistry and an ABI3100 automated sequencer (Applied Biosystems, Foster City, CA). Electropherogram quality was analysed using Chromas® (Technelysium Pty Ltd, South Brisbane, Australia), and methylation patterns were processed using the QUantification tool for Methylation Analysis (QUMA) (Kumaki et al., 2008 (link)). DNA sequences were compared with GenBank reference sequences (Table 2), and only those sequences originating from clones with ≥ 95% identity and ≥ 97% cytosine conversion were used in the analysis (n = 684). The efficiency of the bisulphite treatment was calculated based on the percentage of CpH (H = A, C, or T) site conversion divided by the total number of CpH sites in the sequence.
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2

Recombinant Peptide Expression in B. subtilis

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Plasmid pMK4 as an expression vector used in this B. subtilis expression system was purchased from Addgene (Cambrige, Cambrige, MA, USA). B. subtilis 168 as host cells for recombinant peptide expression was from American Type Culture Collection (ATCC, Manassas, VA, USA). E. coli DH5α and enteropathogenic E. coli 2134P (F18ac) were from our laboratory. Salmonella typhimurium (S. typhimurium), Haemophilus parasuis (H. parasuis), and Staphylococcus aureus (S. aureus) were presented by the Laboratory of Microbiology in College of Food Science and technology.
Restriction enzymes, T 4 DNA ligase and PureYield Plasmid Miniprep were from Promega (Madison, WI, USA). Enterokinase was from Sigma (St. Louis, MO, USA). DL-2000 DNA markers were from Takara Biotechnology (Dalian, China). DNA Purification Kit, Protein Markers were from Transgen Biotechnology (Bejing, China). Mouse anti-His monoclonal antibody and goat anti-mouse IgG-AP antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ni-NTA-agarose beads were from Qiagen (Hilden, Germany).
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3

DNA Amplification and Cloning Protocol

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The primers and oligonucleotides used in the present study were ordered from IDT and are listed in Table 2. DNA was amplified using Phusion HSII polymerase (Thermo), digested with FastDigest restriction enzymes (Thermo), blunted with Fast DNA Repair kit (Thermo), and ligated with Fast-Link DNA ligase (Epicentre). All plasmids were extracted from cells using PureYield plasmid miniprep (Promega), and sequences were verified with Sanger sequencing in the University of Minnesota Genomics Center (UMGC).
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4

Illumina-based Sequencing of Vibrio cholerae S24

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DNA was extracted using the Wizard genomic DNA purification kit (Promega). Plasmid and PCR/gel extractions were done using PureYield Plasmid Miniprep and Wizard SV Gel and PCR clean-up systems respectively (Promega). Purified DNA from V. cholerae S24 was sequenced at the Wellcome Trust Sanger Institute using Illumina-based technology.
All primers used in this study are shown in Table 1. Standard PCR was performed using the PCR master mix (Promega) containing 25 units ml−1 of Taq DNA polymerase, 800 μM dNTPs and 1.5 mM MgCl2. Primers were used at a final concentration of 0.5 μM each. All PCRs were performed with 30 cycles of denaturation at 94°C for 30 s, the appropriate annealing temperature for 30 s and an extension of 72°C (1 min kb−1) and sequencing performed at Macrogen. From whole genome sequencing (Wellcome Trust Sanger Institute) it became evident that the host recA had been disrupted and was present on two separate contigs. The two contigs (contigs 675 and 708) were pieced together by PCR and joined to an intervening third contig to produce a contig of 262,869 bp (contig 367) using primers described in Table 1. The accession number for RME is KJ123688.
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5

TcC Variant Mutagenesis and Expression

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All DNA manipulations described in this work were performed by standard methods.41 Megaprimers for the TcC variants, possessing the specific mutation (Table S1) were purchased from Microsynth AG (Switzerland) and used in two-stage PCR reactions to introduce mutations using Pfu DNA polymerase (Promega GmbH, Germany) and pET26b(+)_TcC as template. Restriction enzymes were purchased from New England Biolabs and used following the manufacturer's protocol. Plasmid DNA was prepared using PureYield™ Plasmid Miniprep and Plasmid Midiprep system (Promega GmbH, Germany). The amplified products were chemically transformed in Escherichia coli XL-10.
For HiC the codon-optimized genes (i.e. E. coli codon usage) of wild-type, I167Q and L64H/I167Q variants were purchased from GeneArt® (Life Technologies, USA). The genes were cloned into the vector pET26b(+) and chemically transformed by heat shock into E. coli XL-10.
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6

Amplification and Sequencing of Mycoplasma obg Gene

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PCR products generated in obg-FR PCR (841-bp) were separated through 1% agarose gels, bands of expected size were excised, purified using Wizard® SV Gel and PCR clean-Up System (Promega) and cloned into pGEM®-T Easy vector (Promega) using instructions provided by the manufacturer. The resultant constructs were propagated in α-select competent cells, silver efficiency (Bioline, Alexandria, New South Wales, Australia) and extracted using PureYield Plasmid Miniprep (Promega). All purified PCR products or plasmid extracts were subjected to automated sequencing (BigDye Terminator v3.1; Applied Biosystems, Foster City, California, USA) in both directions using primers obg-F and obg-R, or M13 forward and reverse sequencing primers for purified PCR products or cloned PCR products, respectively. Nucleotide sequences were edited using SeqMan II and EditSeq programs in DNASTAR. Multiple sequences were aligned using computer program ClustalW2. Nucleotide sequence of complete obg of 25 strains including MS-H and its related strains, belonging to four different genotypes, has been described in our previous study [18] (link). Nucleotide sequence of partial obg of additional 10 M. synoviae strains, including 94041/12a, 4GPH3, F10-2AS, K1938, K870, K1858, YA, K1968, K1723 and WVU-1853, has been submitted to GenBank under accession numbers KF875990 to KF875999.
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7

Plasmid Preparation and Enzymatic Manipulation

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Diepoxybutane (DEB, 11.267M) was purchased from the Sigma Aldrich Company, Inc. (St. Louis, Missouri). Thermo-sensitive alkaline phosphatase (Cat# M9110), and the restriction enzymes Kpn1 (Cat# R6341) and Xho1 (Cat# R6161) were obtained from the Promega Corporation. The T4 ligase DNA Ligation Kit (Catalog #203003) was obtained from Agilent Technologies, Inc. (Santa Clara, California). Both PureYield Plasmid Miniprep (Cat# A1222) and Midiprep Systems (Cat# A2492) were purchased from the Promega Corporation. Quantum Prep® PCR Kleen Spin Columns (Cat# 732–6301) were obtained from Bio-Rad (Hercules, California).
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8

Chickpea PGIPs Cloning and Sequencing

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The total RNA was extracted from the leaves of chickpea cultivar Dwelley using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 100 mg samples in accordance with the manufacturer’s protocol. First strand cDNA synthesis and genomic DNA elimination were performed simultaneously using 5X All-In-One RT MasterMix, containing AccurT Genomic DNA Removal (Applied Biological Materials Inc., Richmond, Canada). cDNA samples were stored at−80 °C until use. Full-length ORFs were amplified with Phusion® High-Fidelity DNA Polymerase (NEB, Ipswich, MA, United States) using gene-specific primer pairs (Supplementary Table S1) using the following protocol: initial denaturation at 98°C for 30 s and 35 cycles of 98°C for 10 s, 60°C and 72°C for 30 s each and followed by a final elongation at 72°C for 8 min. Amplified PCR products with appropriate expected sizes were purified with the Monarch® DNA Gel Extraction Kit (NEB). Purified PCR products were cloned into the pMiniT 2.0 vector (NEB) and transformed into DH10B high-efficiency E. coli competent cells (NEB). The plasmids were recovered from E. coli using PureYield™ Plasmid Miniprep (Promega, Madison, WI, United States), verified using Sanger sequencing (Laboratory of Biotechnology & Bioanalysis, Pullman, WA, United States), and compared to the GenBank sequences of CaPGIP1 (XM_004504675), CaPGIP3 (XM_004493500), and CaPGIP4 (XM_012713804).
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9

Preparation of Lipid Biosynthesis Enzymes

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The genes plsB and plsC were kindly provided by Dr. Yutetsu Kuruma [15 (link)] in the form of circular plasmids. The plasmids carrying an ampicillin selection marker were amplified in E. coli (TOP10) cells and purified with a PureYield plasmid miniprep (Promega). Linear DNA templates were generated by PCR using the primers reported in Table C in S1 File. For the lipid headgroup modification experiments the DNA templates containing the genes cdsA, pgsA, pgpA, pgpC, psd or pssA were all constructed from E. coli MG1655 (K12) genomic DNA that was extracted with GenElute Bacterial Genomic DNA Kit (Sigma Aldrich). The genes were cloned into a pET11a backbone by Gibson assembly [45 (link),46 (link)]. Amplification of the target genes was performed using two primers. A forward primer with the following regions (from 5’ to 3’): a region overlapping the pET11a sequence and a region overlapping the gene of interest. A reverse primer with the same sequence design (pET11a homology, gene of interest homology) was employed. The assembled plasmids were then amplified in E coli cells (TOP10), purified and linear PCR products were generated as described above. All sequences were confirmed by sequencing. The list of primers is reported in Table C in S1 File. Proteins were then synthesized in vitro using either the plasmid or linear DNA as a template for gene expression.
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