The smFRET DNA templates generated by PCR for the toeprinting experiments (see above) were ligated into SmaI-cut pUC18 vector and the resulting plasmids were transformed into E. coli JM109 cells (Promega). Following verification by Sanger sequencing of the constructs, plasmids were cut with EcoRI and used for in vitro run-off transcription with T7 RNA polymerase. Transcription reaction was carried out in the presence of Biotin-GMP to generate 5’ biotinylated RNA.
E coli jm109 cells
Manufactured by Promega
Sourced in United States
E. coli JM109 cells are a commonly used strain of Escherichia coli bacteria. They are considered a general-purpose cloning host suitable for a variety of molecular biology applications.
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Lab products found in correlation
Pgem t easy vector, by Promega (3 mentions)
Ampicillin, by Merck Group (2 mentions)
Pgem t easy kit, by Promega (2 mentions)
Genomic dna clean and concentrate kit, by Zymo Research (2 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Quantitect sybr green pcr master mix, by Qiagen (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Pgl4.23 luc2 minp vector, by Promega (1 mentions)
Xhoi and bglii, by New England Biolabs (1 mentions)
Chocolate agar, by Thermo Fisher Scientific (1 mentions)
Glutathione agarose beads, by GE Healthcare (1 mentions)
Sarkosyl, by Merck Group (1 mentions)
X gal, by Merck Group (1 mentions)
Pgex 2tk vector, by Cytiva (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
15 protocols using e coli jm109 cells
1
Biotinylated RNA Generation from PCR Templates
2
Recombinant Protein Expression in E. coli
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Recombinant PolI_G20c was produced in E. coli Origami (DE3) cells (Novagen) heat-shock transformed with the expression construct. The cells were grown in baffled Erlenmeyer flasks in Lysogeny Broth (Lennox) (LB) medium supplemented with 100 µg ml−1 ampicillin at 37°C. Heterologous overexpression of PolI_G20c was induced with 1 mM isopropyl β-d -1-thiogalactopyranoside when the culture reached an optical density at 600 nm (OD600 nm) of 0.3. Induction was performed for 4 h at 20°C and 200 rev min−1.
Recombinant ExnV1 was produced in E. coli JM109 cells (Promega) heat-shock transformed with the expression construct. The cells were grown in baffled Erlenmeyer flasks in LB medium supplemented with 100 µg ml−1 ampicillin at 37°C. Heterologous overexpression of ExnV1 was induced with 0.2%(w/v)l -rhamnose when the culture reached an OD600 nm of 0.3–0.4. Induction was performed for 4 h at 30°C and 200 rev min−1. Expression cultures were harvested by centrifugation at 5000g for 15 min at 4°C. The collected cell pellets were stored frozen until the purification of the produced recombinant proteins.
Recombinant ExnV1 was produced in E. coli JM109 cells (Promega) heat-shock transformed with the expression construct. The cells were grown in baffled Erlenmeyer flasks in LB medium supplemented with 100 µg ml−1 ampicillin at 37°C. Heterologous overexpression of ExnV1 was induced with 0.2%(w/v)
3
Quantifying Mitochondrial DNA Copy Number
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mtDNA copy number was determined according to our published protocol [13 (link)]. For in vitro model study, the purified PCR product of cytochrome b (Cytb) gene was inserted into pGEM-T easy vector and E. coli JM 109 cells (Promega, Madison, WI, USA) were transformed in order to obtain recombinant plasmids. The mtDNA copy number was calculated using the following formula: [X
μg/μL plasmid DNA/4419 (plasmid length) × 660] × 6.022 × 1023 = Y molecules/μL, where X represents the concentration of plasmid DNA and Y represents copy number. For in vivo model study, a mixture of 25 μL containing 12.5 μL 2x QuantiTect SYBR green PCR master mix (Qiagen, Valencia, CA, USA), 400 μM Cytb primers forward (5′-TTCTGAGGGGCCACAGTAAT-3′) and reverse (5′-GGGGTTATTTGATCCGGTTT-3′), and 50 ng of total DNA were used for the PCR with the CFX96 real-time system (Bio-Rad, Hercules, CA, USA). For PCR, 95°C for 15 minutes was followed by 35 cycles of 20 seconds at 94°C, 30 seconds at 52°C, 30 seconds at 72°C, and a melting reaction with a decrease of 1°C per cycle between 72°C and 92°C.
μg/μL plasmid DNA/4419 (plasmid length) × 660] × 6.022 × 1023 = Y molecules/μL, where X represents the concentration of plasmid DNA and Y represents copy number. For in vivo model study, a mixture of 25 μL containing 12.5 μL 2x QuantiTect SYBR green PCR master mix (Qiagen, Valencia, CA, USA), 400 μM Cytb primers forward (5′-TTCTGAGGGGCCACAGTAAT-3′) and reverse (5′-GGGGTTATTTGATCCGGTTT-3′), and 50 ng of total DNA were used for the PCR with the CFX96 real-time system (Bio-Rad, Hercules, CA, USA). For PCR, 95°C for 15 minutes was followed by 35 cycles of 20 seconds at 94°C, 30 seconds at 52°C, 30 seconds at 72°C, and a melting reaction with a decrease of 1°C per cycle between 72°C and 92°C.
4
Biotinylated RNA Generation from PCR Templates
5
Cloning and Purification of pvceltos
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Purified PCR products for obtaining the pvceltos fragment were ligated into pEXP5-CT/TOPO vector in frame with a His tag at the carboxyl terminal extreme to enable their purification by affinity chromatography and their identification by Western blot, flow cytometry and IFA studies, using anti-polyhistidine Abs. The ligation product was used for transforming E. coli JM109 cells (Promega), following the manufacturer's indications. Recombinant clones were then confirmed by PCR; an UltraClean 6 Minute Mini Plasmid Prep kit (MO BIO) was used for purifying the plasmids and correct insert orientation was evaluated by sequencing, using universal primers.
6
Cloning Luciferase Reporter Construct
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The PCR amplified fragments were digested using XhoI and BglII (New England Biolabs, MA, USA) and ligated into a pGL4.23[luc2/minP] vector (Promega E8411, Madison, MI, USA), previously digested using the same endonucleases. After an overnight incubation at 16°C, the ligation mixtures were transformed into competent E. coli JM109 cells (Promega). Plasmid DNA was isolated by Miniprep kit (Qiagen) according to the manufacturer’s instructions. All recombinant plasmid sequences were confirmed by Sanger sequencing.
7
Microbial Community Identification by Clone Libraries
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We constructed two bacterial clone libraries based on the 16S rRNA gene, and two fungal clone libraries based on the ITS region. PCR amplification of both the 16S rRNA gene and ITS region were performed following the same protocol described for bacterial and fungal PCR-DGGE, respectively, except for the forward primers of the nested PCR. Specifically, for both bacterial and fungal PCR, the forward primers were free of the GC-clamp, and the fungal forward primer (ITS1-F) was complete (5′-CTTGGTCATTTAGAGGAAGTAA) (Gardes and Bruns 1993 (link)).
For each set of early and late incubated eggs, we pooled ten nested PCR products together in order to identify bacterial and fungal communities. Each pool of nested PCR products was ligated into pGEM®-T-Easy vectors (Promega) and introduced into competent E. coli JM 109 cells according to the manufacturer's instructions (Promega). We tested about 20% of the white colonies to estimate cloning efficiency. For each library, 96 different colonies were picked and individually plated on LB agar completed with 100 μg/mL ampicillin in a 96-well microtiterplate. Samples were sequenced by SeqLab (Göttingen, Germany).
For each set of early and late incubated eggs, we pooled ten nested PCR products together in order to identify bacterial and fungal communities. Each pool of nested PCR products was ligated into pGEM®-T-Easy vectors (Promega) and introduced into competent E. coli JM 109 cells according to the manufacturer's instructions (Promega). We tested about 20% of the white colonies to estimate cloning efficiency. For each library, 96 different colonies were picked and individually plated on LB agar completed with 100 μg/mL ampicillin in a 96-well microtiterplate. Samples were sequenced by SeqLab (Göttingen, Germany).
8
Cultivation of Pathogenic Bacteria
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E. coli JM109 cells (Promega) were grown on Lysogeny Broth agar (Oxoid) supplemented where appropriate with 100 µg/ml ampicillin (Sigma). N. meningitidis serogroup B strain MC58 (ATCC® BAA335™) [49] and clinical isolates of N. meningitidis from our laboratory collection, N. gonorrhoeae strain FA1090 (ATCC 700825), N. lactamica strain ATCC23970, N. polysaccharea (a clinical isolate from laboratory stocks), H. influenzae Rd KW20 (ATCC 51097) [50] , and S. pneumoniae T4 (unencapsulated) [51] were routinely cultured on chocolated horse blood agar (Chocolate agar, Oxoid) at 37°C, in an atmosphere of air plus 5% CO2.
9
Mycobacterium tuberculosis Protein Purification
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Blood sample as source local strain of M. tuberculosis was collected from the pulmonary tuberculosis patient from the Wahidin Sudirohusodo Hospital, Makassar, Indonesia. Signed written informed consent was obtained according to Ethics Committee from Hasanuddin University Hospital, Makassar, Indonesia. pGEX-2TK vector and E. coli BL21 component cells were purchased from Amersham Pharmacia Biotech. pGEM-T Easy vector and E. coli JM109 cells was purchased from Promega, USA. Glutathione-agarose beads was purchased from GE Healthcare Hong Kong. Ampicillin, IPTG, X-gal, phenylbenzosulfonyl fluoride, dithiothreitol, Sarkosyl, and lysozyme were purchased from Sigma.
10
Heterologous Expression of Bradykinin 2 Receptor in Xenopus Oocytes
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For heterologous expression of bradykinin 2 receptor (B2R in pcDNA3.1+) in Xenopus laevis oocytes, the cDNA fragment was subcloned into a high expression vector pGEM-HE which contains 3’ and 5’ untranslated regions of a Xenopus globin gene (11 (link)). The B2R insert was generated by a double analytic digest with XbaI and BamHI, two restriction sites that flanked the insert. Sticky ends in the vector pGEM-HE were made by cleaving the restriction sites with XbaI and BamHI. Following, the B2R insert was ligated into the linearized pGEM-HE construct with T4 ligase (Thermo Fisher Scientific, USA). After ligation, competent E. coli JM109 cells (Promega, USA) were transformed to express the corresponding cDNA. Next, the cDNA was linearized with XbaI and cRNA was transcribed with a T7 mMESSAGEmMACHINE transcription kit (Ambion, Austin, TX, USA).
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