S. putrefaciens S. putrefaciens 200 used for total DNA extraction was provided to us as a gift from Flynn Picardal, Indiana University. Expression vector pET26b containing S. aureus SH1000 (WP_000358012.1) MurA was kindly provided by Dr. Alex O’Neill (University of Leeds, Leeds, UK). E. coli One Shot™ TOP10 (Thermo Fisher Scientific, Waltham, MA) (F-mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 Δ lacX74 recA1 araD139 Δ(araleu)7697 galU galK rpsL (StrR) endA1 nupG) was used for library construction and in vivo arsenical resistance assays. E. coli strain BL21 (fhuA2 [lon] ompT gal (λ DE3) [dcm] ΔhsdS λ DE3 = λ sBamHIo ΔEcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 Δnin5) was used for expression and purification of S.putrefaciens 200 wild-type and C117D MurA, as well as in vivo fosfomycin resistance assays. E. coli strains were grown aerobically at either 30 °C or 37 °C in either lysogeny broth (LB) or 2x ST medium (20-fold concentrated ST 10−1 media, 10 g L−1 Difco Bacto Peptone and 1 g L−1 yeast extract) supplemented with 0.2% glucose (Maki et al., 2006 ).
One shot top10
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The One Shot TOP10 is a chemically competent E. coli strain designed for high-efficiency transformation of plasmid DNA. It is optimized for blue/white screening and DNA manipulation.
Automatically generated - may contain errors
Lab products found in correlation
Qiaprep spin miniprep kit, by Qiagen (4 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Miniprep kit, by Zymo Research (2 mentions)
Gateway lr clonase 2, by Thermo Fisher Scientific (2 mentions)
Pentr directional topo vector, by Thermo Fisher Scientific (2 mentions)
Pfuultra 2 fusion polymerase, by Agilent Technologies (2 mentions)
Abi 3130 capillary sequencer, by Thermo Fisher Scientific (2 mentions)
Bl21 de3, by New England Biolabs (2 mentions)
Topo ta, by Thermo Fisher Scientific (2 mentions)
Luria bertani medium, by Thermo Fisher Scientific (2 mentions)
Pcdna3.1 v5 his topo cloning vector, by Thermo Fisher Scientific (2 mentions)
Pgem t easy vector system, by Promega (2 mentions)
Abi 3730xi dna analyzer, by Thermo Fisher Scientific (2 mentions)
Zero blunt topo cloning kit, by Thermo Fisher Scientific (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Peak scanner, by Thermo Fisher Scientific (1 mentions)
Quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Max efficiency dh5α, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
61 protocols using one shot top10
1
MurA Protein Expression and Purification
2
Androgen Receptor and MECP2 Allele Analysis
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Androgen receptor (AR) assay was performed as previously described [10 (link)]. In brief, 400 ng of genomic DNA from iPSCs/ESCs was digested by CpG methylation-sensitive restriction enzymes HpaII and HhaI for 6 h. 2 μL of digestion was amplified using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with AR gene primers with a FAM label (Table S2 ). Male samples were used as a control to confirm complete digestion. Electrophoresis was performed by TCAG at The Hospital for Sick Children. Traces were analyzed using PeakScanner (Thermo Fisher Scientific). To identify which MECP2 allele was expressed on the active X chromosome, RNA was isolated from iPSCs and reverse transcribed into cDNA using SuperScript III (Invitrogen). Primers flanking the variant region (Table S2 ) were used for amplification with Platinum Taq DNA Polymerase High Fidelity or Q5 High Fidelity DNA Polymerase (New England BioLabs), followed by cloning as per the TOPO TA Cloning Kit (Invitrogen) or Zero Blunt TOPO Cloning Kit (Invitrogen) in OneShot TOP10 (Thermo Fisher Scientific) or Max Efficiency DH5α (Invitrogen) competent E. coli strains. 5–10 bacterial colonies per iPSC cell line tested were picked and grown in LB Medium (MP Biomedicals) for DNA extraction with Quick Plasmid MiniPrep Kit (Invitrogen). Samples were Sanger sequenced at TCAG and aligned to WT MECP2 template using benchling.com.
3
RPGRIP1 Exon Cloning and Splicing Assay
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PCR was performed using PfuUltra II Fusion polymerase (Agilent Technologies, Santa Clara, CA) on genomic DNA of patients harboring the mutations of interest (primers are listed in the Supplementary ). The PCR products were cloned into pENTR Directional TOPO vector (Thermo Fisher, Waltham, MA) and used to transform chemically competent Escherichia coli (One Shot TOP10, Thermo Fisher, Waltham, MA). Plasmid DNA from single colonies was extracted with miniprep kits (ZymoPURE, Zymo Research) and analyzed by restriction enzyme digestion with BsrGI (NE Biolabs, Ipswich, MA) and Sanger sequencing. Essential splice-site mutations were introduced by site-directed mutagenesis (QuickChange II Site Directed mutagenesis kit, Agilent Technologies) and verified by Sanger sequencing. Colonies with the correct sequence and restriction enzyme pattern were then sub-cloned into the pCS2+GW vector (kind gift from Dr Erica Davis) via Gateway LR clonase II (Thermo Fisher) and similar analyses as before was done to isolate vectors with the appropriate inserts for transfection experiments. The final vector included RPGRIP1 exons 11–16 including extensions into intron 10 and 16 on the 5’ and 3’ ends, which was cloned into pCS2+GW and used for splicing assays.
4
Detailed Molecular Cloning Protocol
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All PCR amplifications were performed using Q5® DNA Polymerase (New England Biolabs) according to the manufacturer’s instruction. All GG cut-ligation reactions were performed according to the described protocol (Additional file 1 : section 1).
All ligations were transformed into One Shot™ TOP10 chemically competent E. coli (Thermo Fisher Scientific) and constructs were verified by sequencing (Eurofins Genomics).
Specific details related to assembly of all GG modules reported in this study are provided (Additional file1 : section 4). Sequences of all PCR primers used in the study can be found in Table S4 (Additional file 1 ). All DNA constructs generated as part of this study were deposited with Addgene (www.addgene.org ) with Addgene IDs indicated for each construct (Additional file 2 : Table S5). The toolkit comprising 95 constructs will also be available from Addgene in a 96-well plate under the reference number 1000000159. The rest of the materials (8 out of 103 constructs) can be requested by contacting the corresponding author. Sequence information of all constructs can be found in GenBank (.gb) files at www.addgene.org and on Figshare (10.6084/m9.figshare.11961975).
All ligations were transformed into One Shot™ TOP10 chemically competent E. coli (Thermo Fisher Scientific) and constructs were verified by sequencing (Eurofins Genomics).
Specific details related to assembly of all GG modules reported in this study are provided (Additional file
5
RPGRIP1 Exon Cloning and Splicing Assay
6
Sanger Sequencing of Amplified Products
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For Sanger sequencing of the amplification products, bands of the expected size were excised from agarose gels and purified with a commercial kit (NucleoSpin® Gel and PCR Clean-up; Macherey–Nagel, Düren, Germany), following the manufacturer’s instructions. Purified amplification products were then cloned into a commercially available vector (pGEM®-T Easy Vector System I; Promega, Mannheim, Germany) and used to transform chemically competent Escherichia coli (OneShot TOP10; Thermo Fisher Scientific, Langenselbold, Germany). The transformed E. coli cell were cultivated and the plasmid DNA was subsequently collected using a commercial kit (QIAprep Spin Miniprep Kit; Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Sequencing was performed using the BigDye Terminator v1.1 Cycle Seq. Kit (Thermo Fisher Scientific) and passage through NucleoSEQ Columns (Macherey–Nagel) for cleaning up the nucleic acids, in an ABI 3130 capillary sequencer (Thermo Fisher Scientific).
The forward and reverse sequences were aligned, if necessary trimmed based on primer sequence information, and the consensus sequences for the individual cloned amplification products compared to sequences stored in GenBank, EMBL, DDBJ or RefSeq using BLASTn with standard conditions.
The forward and reverse sequences were aligned, if necessary trimmed based on primer sequence information, and the consensus sequences for the individual cloned amplification products compared to sequences stored in GenBank, EMBL, DDBJ or RefSeq using BLASTn with standard conditions.
7
Transformation of Chemically Competent E. coli
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50 μl ONE Shot TOP10 or ONE SHOT Stbl3 Chemically Competent E.coli (Thermo Fisher Scientific) were thawed on ice for each transformation. 5 μl plasmid DNA was added to the competent cells and mixed by flicking the tube before storing on ice for 30 min. Cells were then placed at 42°C for 30 seconds to enable incorporation of DNA and then placed on ice for 2 min. 250 μl of Super Optimal broth with Catabolite repression (SOC) medium at room temperature was added to each vial and placed in a 37°C orbital shaker for 2 h. After incubation, 20 μl and 200 μl of each transformation was placed on a pre-warmed agar plate containing the appropriate antibiotic and spread using ColiRoller Plating Beads (Millipore). Plates were wrapped in Parafilm (Thermo Fisher), inverted and placed in a 37°C incubator overnight. Plates were checked the next day for bacterial colonies and placed at 4°C until required.
8
Actin Cytoskeleton Modulation in E. histolytica
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The E. histolytica strain HM1:IMSS was cultured in TYI-S-33 medium at 37°C (Diamond et al., 1978 (link)). Drug treatments included latrunculin B (100 nM, for 15 min; this compound binds to actin monomers and prevents actin polymerization), jasplakinolide (Sigma-Aldrich, USA, at 10 μM, for 30 min; this is a filament polymerizing and stabilizing agent), and 2-fluoro-N-[2-(2-methyl-1H-indol-3-yl)ethyl]-benzamide (CK-666, Sigma-Aldrich, USA at 40 μM for 2 h; this compound binds to the Arp2/3 complex, stabilizes it in an inactive state, and prevents the formation of the active conformation). The bacterium Escherichia coli (One Shot TOP10, Thermo Fisher Scientific, USA) was grown in Luria-Bertani medium supplemented with ampicillin (100 μg/ml) and used for plasmids amplification.
9
DNA Extraction and Plasmid Preparation Protocol
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DNA from the vaginal specimens was extracted according to validated in-house laboratory protocols using QIAamp ® DNA Mini Kit (QIAGEN, Maryland, USA) and the X-tractor Gene ® DNA workstation (QIAGEN, Maryland, USA) with slight modifications. Canine Herpes Virus DNA was spiked into the samples and subsequently detected as an internal extraction control.
To serve as amplification standards, plasmids for each of the 22 bacterial species were generated by whole-gene synthesis (Genewiz ® , Azenta Life Sciences, Waltham, USA). Briefly, a species-specific region of each species was amplified, synthesized, and cloned into the pUC-GW-AMP plasmid vector with an ampicillin-resistance marker. These plasmids were transformed into chemically competent Escherichia coli cells (One Shot ™ TOP10, ThermoFisher Scientific, New Jersey, USA) and grown in liquid Lysogeny Broth (LB) containing 50 µg/mL Ampicillin. Plasmid DNA was isolated from overnight cultures using Wizard ® Plus SV Minipreps DNA Purification Systems (Promega, Wisconsin, USA) according to the manufacturer's protocol. Extracted DNA was subsequently quantified spectrophotometrically using NanoDrop 1000 equipment (ThermoFisher Scientific, New Jersey, USA). Standard 10-fold serial dilutions of 10 8 to 10 1 DNA copies/µL were prepared for each bacterial species.
To serve as amplification standards, plasmids for each of the 22 bacterial species were generated by whole-gene synthesis (Genewiz ® , Azenta Life Sciences, Waltham, USA). Briefly, a species-specific region of each species was amplified, synthesized, and cloned into the pUC-GW-AMP plasmid vector with an ampicillin-resistance marker. These plasmids were transformed into chemically competent Escherichia coli cells (One Shot ™ TOP10, ThermoFisher Scientific, New Jersey, USA) and grown in liquid Lysogeny Broth (LB) containing 50 µg/mL Ampicillin. Plasmid DNA was isolated from overnight cultures using Wizard ® Plus SV Minipreps DNA Purification Systems (Promega, Wisconsin, USA) according to the manufacturer's protocol. Extracted DNA was subsequently quantified spectrophotometrically using NanoDrop 1000 equipment (ThermoFisher Scientific, New Jersey, USA). Standard 10-fold serial dilutions of 10 8 to 10 1 DNA copies/µL were prepared for each bacterial species.
10
Overexpression and Knockdown of OPN Gene
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OPN-c gene of pcDNA3-OPN-V5 was a gift from Steven Johnson (Addgene plasmid # 11617). It was amplified by Phusion HF DNA polymerase (M0530L) and purified by the QIAquick PCR Purification Kit (#28106) and QIA gel Extraction kit (#28706). The PCR product was cut with NEB CutSmart and ligated overnight at 16ºC to p3XFLAG-CMV™-7.1 expression vector (E7533 SIGMA). Transformation was performed with competent E. coli cells (One Shot Top10, #C404003, Thermofisher). The overexpression gene transient transfection was carried out with Lipofectamine 3000 reagent (Invitrogen, CA, USA), while transient knockdown using Mission® esiRNA (EHU018231 SIGMA) with Lipofectamine 2000 following the manufacturer's protocol. The cell was seeded to be 60-70% confluent prior transfection. The ratio of plasmid DNA/Lipofectamine 3000 was selected as 2.5 μg / 3.75 μl for the OPN overexpression transfection and siRNA/Lipofectamine 2000 for transient knockdown was used at 1200 ng / 4 μl process (5× 105 cells in 3 ml complete culture medium per well of a 6-well plate). The cell was post-transfected for 48 hours at 37°C, then the transfection efficacy was determined by qPCR quantification and western blot analysis.
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