V. cholerae mutants used in this study were derived from El Tor biotype strain C7258 and are described in Table S3 . Escherichia coli TOP10 (Life Technologies) and S17-1λpir (de Lorenzo et al., 1993 (link)) were used for cloning purposes. V. cholerae strains were grown in LB medium at 37°C with agitation (225 rpm). When necessary, culture media were supplemented with ampicillin (Amp) (100 μg/ml), chloramphenicol (Cm) (10 μg/ml), kanamycin (Km) (25 μg/ml), rifampin (Rf) (150 μg/ ml), polymyxin B (PolB) (100 units/ml), isopropyl-β-D-thiogalactopyranoside (IPTG) (0.05 to 1.0 mM as indicated), or 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) (20 μg/ml). Plasmids and oligonucleotide primers used throughout this work are described in Tables S3 and S4 , respectively.
Escherichia coli top10
Manufactured by Thermo Fisher Scientific
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Escherichia coli TOP10 is a laboratory strain of the bacterium Escherichia coli. It is commonly used in molecular biology and genetic engineering applications for the cloning and amplification of DNA constructs.
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Kanamycin, by Merck Group (7 mentions)
Chloramphenicol, by Merck Group (6 mentions)
Restriction enzyme, by New England Biolabs (4 mentions)
E coli dh5α, by Thermo Fisher Scientific (3 mentions)
Luria bertani medium, by BD (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Superscript 3 first strand synthesis kit, by Thermo Fisher Scientific (2 mentions)
Qiaprep kit, by Qiagen (2 mentions)
Bamhi, by New England Biolabs (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Gel purified, by Zymo Research (2 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (2 mentions)
E coli bl21 rosetta 2, by Merck Group (2 mentions)
Ppiczαa, by Thermo Fisher Scientific (2 mentions)
Xylan, by Merck Group (2 mentions)
P pastoris gs115, by Thermo Fisher Scientific (2 mentions)
Ppic3.5k, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Takara Bio (2 mentions)
Erythromycin, by Merck Group (2 mentions)
Hygromycin, by Thermo Fisher Scientific (2 mentions)
151 protocols using escherichia coli top10
1
Vibrio cholerae Mutant Strains and Cloning
2
Antimicrobial Activity Assay of C3
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We tested the antimicrobial activity of C3 through a series of disk diffusion assays (Bhunia et al., 1987 (link)). Using a hole punch, we made 6 mm disks from Whatman filter paper which were sterilized in the autoclave. Overnight cultures of our indicator strains (see below) were grown in nutrient broth incubated at 37°C while shaking (225 rpm). The overnight indicator strain liquid was added to molten soft agar (0.7%) at 1:100 v/v. The agar was gently mixed, poured into sterile petri dishes in 10 mL volumes, then allowed to cool for 30 mins. The C3 strain was prepared by mixing an isolated colony in LB liquid media and incubating overnight at 37°C shaking (225 rpm). The C3 liquid cultures were centrifuged at 1,791 × g for 10 min and 25 μL of the supernatant was removed and applied to the filter disks placed on the surface of soft nutrient agar plates previously inoculated with a competing species. Bacterial strains used in antimicrobial activity assays include Escherichia coli TOP10 (Life Technologies, Grand Island, NY), Pseudomonas syringae (kindly provided by R. Innes, Indiana University), Xanthomonas axonopodis pathovar Starr and Garces pathovar phaseoli (ATCC 9563), Bacillus subtilis (ATCC 6051). Plates were incubated at 37°C for 48–72 h followed by inspection of the zone of inhibition.
3
Cultivation and Identification of Candida utilis
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C. utilis wild-type strain DSMZ2361 (ATCC9950), obtained from Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany) was used in this study. C. utilis, also known as Torula yeast has been classified as a (generally recognized as safe) organism by the Food and Drug administration (FDA; http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/MicroorganismsMicrobialDerivedIngredients/default.htm ). Strain MKCu1 was used as control [19 (link)]. Yeast strains were grown in YPD media (1% yeast extract, 2% peptone and 2% glucose) at 30°C on a horizontal shaker (110 rpm). To select C. utilis transformants media were supplemented with 10 μg/ml Nourseothricin (NST; Jena Bioscience, Jena, Germany). For identification of C. utilis cells in fecal pellets, the pellets were resuspended in PBS and plated out in a serial dilution on agar plates supplemented with 10 μg/ml NST and 100 μg/ml ampicillin (life technologies, Darmstadt, Germany) to prevent bacterial growth. Escherichia coli TOP10' (life technologies) cells were used for plasmid construction and were grown in LB media at 37°C supplemented with 100 μg/ml of ampicillin.
4
Heterologous Expression of CYP Genes
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Libraries of cDNA were synthesized from the same RNA samples used for RNA-seq using SuperScript III First-Strand Synthesis kit (Life Technologies) according to the manufacturer’s instructions. CYP-encoding sequences were amplified from the cDNA library by PCR using Phusion DNA Polymerase (Thermo Scientific) and oligonucleotide primers (Integrated DNA Technologies; sequences given in Supplementary Table 5 ). PCR products of the desired size were gel-purified (Zymo Research), inserted into the vector pYeDP60 (ref. 21 (link)) using the BamHI and EcoRI restriction sites (enzymes from New England Biolabs), and transformed into Escherichia coli TOP10 (Life Technologies). Because RCBr is heterozygous at some genomic loci, eight individual clones of each CYP were purified (QIAprep kit from Qiagen), sequenced (Elim BioPharm), and compared. If two alleles for a particular CYP were present, then the allele with the higher % amino acid identity to the B. rapa Chiifu reference genome was chosen. The procedure for heterologous expression of CYPs using Saccharomyces cerevisiae strain WAT11 (kindly supplied by Dr. Franck Pinot)21 (link) was identical to that described in Klein et al.20
5
Cultivation of the Marine Diatom F. solaris
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The marine pennate diatom F. solaris JPCC DA0580 cells were maintained in f/2 medium [41 (link)] dissolved in artificial seawater. The transformed cells were cultivated in the same medium with the addition of antibiotics G-418 (500 μg/ml). Cultures (500 ml of f/2 medium in flat-shaped flasks) were incubated at 25 °C under continuous, cool-white fluorescent lights at 140 μmol/m2/s with aeration (0.8 l of air containing 2 % CO2/l/min). Genes were cloned in Escherichia coli TOP10 (Life Technologies, Tokyo, Japan) cultured in a lysogeny broth (LB) medium containing 50 μg/ml ampicillin at 37 °C.
6
Heterologous Expression of CYP Genes
7
Bacterial Transformation and Plasmid Cloning
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Example 1
Chemical competent Escherichia coli TOP10 (Life Technologies; US) and BL21(DE3) Gold (Agilent Technologies; Germany) was used a recipient in transformation experiments. Transformation was done as described by Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Habor Laboratory Press, Cold Spring Habor; N.Y. (1982). Agrobacterium tumefaciens was used to introduce the T-DNA region into Arabidopsis, corn and soybean.
Bacterial cultures were routinely grown on Luria broth (LB) or at 37° C. on LB mixed with agar (15% w/v). LB was also supplemented with antibiotic kanamycin and/or chloramphenicol where required. Plasmid DNA was prepared using GeneJet Plasmid Miniprep kit (Thermo Scientific, US). TriA and variants of thereof were generated by gene synthesis (Eurofins, Germany). Synthesized genes harboring XhoI and NcoI restriction sites were cloned into pET24d N-HIS vector with kanamycin resistance. Chaperone plasmid pGro7 (chaperones groEL and groES) with chloramphenicol resistance was obtained from TaKaRa (Japan).
8
Cultivation of Phaeodactylum and E. coli
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A reference strain of Phaeodactylum (CCAP-1055/1) was used in all experiments. Phaeodactylum was grown at 18°C under white fluorescent lights (50 uE m–2 s–1) and subjected to a diel growth cycle (14 h:10 h; light:dark). Culture medium was artificial sea water (ASW) supplemented with trace metals, essential vitamins, 55 uM of NaPO4, and 880 uM of the appropriate nitrogen source (NaNO3, NH4Cl, or Urea). Cultures, when not used in growth assays, were maintained with chloramphenicol antibiotic (10 mg/L) to keep cultures bacteria-free. Mutant Phaeodactylum strains were supplemented with either phleomycin (50 mg/L), zeocin (50 mg/L), or nourseothricin (200 mg/L).
Escherichia coli (TOP-10, Life Technologies, Carlsbad, CA) was used for all molecular cloning purposes. The cultures were grown on Luria-Bertani broth or agar and supplemented with the following antibiotics when necessary: ampicillin (100 mg/L), carbicillin (100 mg/L), tetracyclin (10 mg/L), gentamicin (20 mg/L), zeocin (25 mg/L).
Escherichia coli (TOP-10, Life Technologies, Carlsbad, CA) was used for all molecular cloning purposes. The cultures were grown on Luria-Bertani broth or agar and supplemented with the following antibiotics when necessary: ampicillin (100 mg/L), carbicillin (100 mg/L), tetracyclin (10 mg/L), gentamicin (20 mg/L), zeocin (25 mg/L).
9
Cloning of Antigen Genes
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pQE31 vector (Qiagen GmbH, Hilden, Germany) was used for cloning of antigen genes. Escherichia coli TOP10 (Life Technologies Inc., New York, USA) and E. coli M15 (pREP4) (Qiagen GmbH, Hilden, Germany) were used as intermediate and expression hosts, respectively. Luria Bertani (LB) broth and agar were used for routine culture of E. coli strains. When required, ampicillin (Amp) and kanamycin (Km) were added to culture media at final concentrations of 100 and 25 μg/ml, respectively.
10
Synechocystis salina Lipid Feeding Protocol
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pET-51b was acquired from Novagen. Escherichia coli TOP10 (Life Technologies) was used for cloning and E. coli BL21 DE3 Rosetta (Novagen) was used for recombinant protein expression.
The cyanobacterium Synechocystis salina LEGE 06099 was obtained from the LEGEcc30 (link). Cultures were grown in Z8 medium supplemented with 25 g L−1 sea salt (Tropic Marin), at 25 °C, under a 14:10 h light/dark cycle and constant aeration14 (link). For the feeding studies with 5-hexynoic and 6-heptynoic acids, small-scale cultures were inoculated to a final OD750 of ~0.04–0.1 while large scale cultures (20 L) were inoculated using 1.5 L of stationary phase cultures. Feeding experiments with other fatty acids used a 3:50 inoculum of stationary phase cultures.
The cyanobacterium Synechocystis salina LEGE 06099 was obtained from the LEGEcc30 (link). Cultures were grown in Z8 medium supplemented with 25 g L−1 sea salt (Tropic Marin), at 25 °C, under a 14:10 h light/dark cycle and constant aeration14 (link). For the feeding studies with 5-hexynoic and 6-heptynoic acids, small-scale cultures were inoculated to a final OD750 of ~0.04–0.1 while large scale cultures (20 L) were inoculated using 1.5 L of stationary phase cultures. Feeding experiments with other fatty acids used a 3:50 inoculum of stationary phase cultures.
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