In all measurements, E. coli strain AJ5 is used. The strain is created from strain BW25113 (Datsenko and Wanner, 2000 (link)) by P1 transduction with lysate from strain FW1401 (Wu et al., 2015 (link)). The resulting strain carries a tagRFP-T sequence together with kanamycin resistance cassette replacing leuB. For an experiment, a colony from LB plate is grown overnight at 28°C with shaking in M9 minimal medium. The medium consists of M9 salts (Teknova, CA), supplemented with 2 mM magnesium sulfate (MgSO4), 0.5% (w/v) glucose (Sigma-Aldrich, MO), 0.2% casamino acids (ACROS Organics, NJ). Liquid media are supplemented with 25 μg/ml kanamycin (Sigma-Aldrich, MO) for selection.
Casamino acid
Manufactured by Thermo Fisher Scientific
Sourced in United States, Macao
Casamino acids are a complex mixture of amino acids derived from the enzymatic hydrolysis of casein, a protein found in milk. They serve as a source of nitrogen, carbon, and other essential nutrients for microbial growth and development in various cell culture and microbiological applications.
Automatically generated - may contain errors
Lab products found in correlation
D glucose, by Merck Group (5 mentions)
Thiamine hydrochloride, by Merck Group (5 mentions)
Cacl2, by Merck Group (4 mentions)
Kanamycin, by Merck Group (3 mentions)
Mgso4, by Merck Group (3 mentions)
Spectinomycin, by GoldBio (2 mentions)
Anhydrotetracycline hydrochloride atc, by Merck Group (2 mentions)
L arabinose ara, by Merck Group (2 mentions)
Ampicillin, by Thermo Fisher Scientific (2 mentions)
L arabinose, by Merck Group (2 mentions)
M9 minimal media, by Merck Group (2 mentions)
Mgso4, by Thermo Fisher Scientific (2 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Yeast extract, by Thermo Fisher Scientific (2 mentions)
Yeast extract, by Merck Group (2 mentions)
Egg yolk emulsion, by Merck Group (2 mentions)
Unsheared salmon sperm dna, by Merck Group (2 mentions)
Bordet gengou agar, by BD (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
37 protocols using casamino acid
1
E. coli AJ5 Strain Characterization
2
Engineered E. coli Strain for Expression
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The Escherichia coli DH10B derivative NEB 10-beta Δ(ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14- ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr-hsdRMS-mcrBC) was used as the host (New England Biolabs, MA, C3019). Cells were grown in M9 minimal media, consisting of 1× M9 media salts (Sigma–Aldrich, MO, M6030), 0.34 g/L thiamine hydrochloride (Sigma–Aldrich, MO, T4625), 0.4% D-glucose (Sigma–Aldrich, MO, G8270), 0.2% Casamino acids (Acros, NJ, AC61204-5000), 2 mM MgSO4 (Sigma–Aldrich, MO, 230391), and 0.1 mM CaCl2 (Sigma–Aldrich, MO, 449709). The inducers used in this study were isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma–Aldrich, MO, I6758), anhydrotetracycline hydrochloride (aTc; Sigma–Aldrich, MO, 37919), and L-arabinose (Ara; Sigma–Aldrich, MO, A3256). Antibiotic selections were performed with 50 µg/ml kanamycin (Gold Biotechnology, MO, K-120-5) and 50 µg/ml spectinomycin (Gold Biotechnology, MO, S-140-5). Phosphate buffered saline (1x PBS) solution was prepared from a 10x solution purchased from OmniPur (MA, 6505).
3
Preparation of Artificial Sputum Media for B. cenocepacia
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ASMDM was prepared in 30 mL aliquots as described by Fung et al. [14 (link)]. Porcine stomach mucin (Sigma-Aldrich, St. Louis, MO), DNA from salmon testes (Sigma-Aldrich, St. Louis, MO), potassium chloride (Fisher Scientific, Hanover Park, IL), sodium chloride (Fisher Scientific, Hanover Park, IL), diethylene triamine pentaacetic acid (Fluka Analytical, St. Louis, MO), casamino acids (Acros, Hanover Park, IL), and bovine serum albumin (Sigma-Aldrich, St. Louis, MO) were dissolved in 25 mL sterile dH2O (final concentrations: 10 mg/mL, 1.4 mg/mL, 2.2 mg/mL, 5 mg/mL, 5.9 mg/mL, 5 mg/mL, and 10 mg/mL, respectively). Egg yolk emulsion (0.15 mL) (Becton Dickinson) and the antibiotics ampicillin (1 ug/mL; (Fisher Scientific, Hanover Park, IL) and penicillin (1 ug/mL; Sigma-Aldrich, St. Louis, MO) were added to revent contamination. Tetracycline was omitted because B. cenocepacia is sensitive to this antibiotic [18 (link)]. The suspension was stirred for 5 min at room temperature to dissolve the DNA and mucin. The pH was adjusted to 6.5, and sterile dH2O was added to 30 mL ASMDM was stored at 4 °C, but warmed to 37 °C before addition to cells.
4
Engineered Bacterial Sensor Circuits
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E. coli Nissle 1917 was used for experimentally assaying sensors, genetic
gates, and circuits. E. coli NEB
5-alpha (New England Biolabs) was used for cloning. E. coli NEB 10-beta (New England Biolabs) was
used to compare sensor and NOT gate response functions to EcN. Genetic
circuits and sensors were assayed in M9 minimal media (Sigma-Aldrich;
6.78 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl final concentration)
with 0.34 g/L thiamine hydrochloride (Sigma-Aldrich), 0.2% Casamino
acids (Acros), 2 mM MgSO4 (Sigma-Aldrich), 0.1 mM CaCl2 (Sigma-Aldrich), and 0.4%d -glucose (Sigma-Aldrich).
Antibiotics used to select for circuit plasmids were 50 μg/mL
kanamycin (GoldBio), and 100 μg/mL spectinomycin (GoldBio).
The inputs used for the sensor promoters were isopropyl β-d -1-thiogalactopyranoside (GoldBio), anhydrotetracycline hydrochloride
(Sigma-Aldrich),l -arabinose (Sigma-Aldrich), choline chloride
(Sigma-Aldrich), gamma-aminobutyric acid (Sigma-Aldrich), N-butyryl-dl -homoserine lactone (Sigma-Aldrich),
3-oxohexanoyl-l -homoserine lactone (Sigma-Aldrich), N-(3-oxododecanoyl)-l -homoserine lactone (Sigma-Aldrich),
and N-(3-hydroxytetradecanoyl)-dl -homoserine
lactone (Sigma-Aldrich). The stock solutions were aqueous solutions,
except those used for HSLs and aTc. The stock solutions for HSLs were
dissolved in 100% dimethyl sulfoxide, and aTc was dissolved in 100%
ethanol.
gates, and circuits. E. coli NEB
5-alpha (New England Biolabs) was used for cloning. E. coli NEB 10-beta (New England Biolabs) was
used to compare sensor and NOT gate response functions to EcN. Genetic
circuits and sensors were assayed in M9 minimal media (Sigma-Aldrich;
6.78 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl final concentration)
with 0.34 g/L thiamine hydrochloride (Sigma-Aldrich), 0.2% Casamino
acids (Acros), 2 mM MgSO4 (Sigma-Aldrich), 0.1 mM CaCl2 (Sigma-Aldrich), and 0.4%
Antibiotics used to select for circuit plasmids were 50 μg/mL
kanamycin (GoldBio), and 100 μg/mL spectinomycin (GoldBio).
The inputs used for the sensor promoters were isopropyl β-
(Sigma-Aldrich),
(Sigma-Aldrich), gamma-aminobutyric acid (Sigma-Aldrich), N-butyryl-
3-oxohexanoyl-
and N-(3-hydroxytetradecanoyl)-
lactone (Sigma-Aldrich). The stock solutions were aqueous solutions,
except those used for HSLs and aTc. The stock solutions for HSLs were
dissolved in 100% dimethyl sulfoxide, and aTc was dissolved in 100%
ethanol.
5
Cloning and Characterization of Synthetic Devices
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Cloning was performed using E. coli strain DH5-α (F–endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA–argF)U169, hsdR17(rK–mK+), λ–) (New England Biolabs, C2987I). Device characterization was performed using BL21 Star (DE3) (F–ompT hsdSB (rB–, mB–) gal dcm rne-131 [DE3]) (Thermo Fisher Scientific, C601003). For cloning, cells were grown in LB Miller broth (Sigma-Aldrich, L3522). For device characterization, cells were grown in M9 minimal media supplemented with glucose containing M9 salts (6.78 g/L Na2HPO4, 3 g/L KH2PO4, 1 g/L NH4Cl, 0.5 g/L NaCl) (Sigma-Aldrich, M6030), 0.34 g/L thiamine hydrochloride (Sigma T4625), 0.4% D-glucose (Sigma-Aldrich, G7528), 0.2% casamino acids (Acros, AC61204-5000), 2 mM MgSO4 (Acros, 213115000), and 0.1 mM CaCl2 (Sigma-Aldrich, C8106). Antibiotic selection was performed using 50 μg/mL kanamycin (Sigma-Aldrich, K1637) or 50 mg/mL spectinomycin (Santa Cruz Biotechnology, sc-203279). Induction of sensor systems was performed using aTc (Sigma-Aldrich, 37919), IPTG (Sigma-Aldrich, I6758), and l -Arabinose (Ara) (Sigma-Aldrich, A3256).
6
Growth of E. coli Strains with FtsZ Fusions
7
Experimental Measurements of Genetic Circuits
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Strains, media, and inducers E. coli NEB 10-beta [D(ara-leu) 7697 araD139 fhuA DlacX74 galK16 galE15 e14-f80dlacZDM15 recA1 relA1 endA1 nupG rpsL (Str R ) rph spoT1 D(mrr-hsdRMS-mcrBC)], which is a derivative of E. coli DH10B (85), was used for experimentally measuring circuits. E. coli was cultured in LB Miller medium (Sigma-Aldrich, L3152) for routine cloning and propagation of plasmids. Genetic circuits were assayed in M9 minimal media (Sigma-Aldrich, M6030; 6.78 g/liter Na 2 HPO 4 , 3 g/liter KH 2 PO 4 , 1 g/liter NH 4 Cl, 0.5 g/liter NaCl final concentration) with 0.34 g/liter thiamine hydrochloride (Sigma-Aldrich, T4625), 0.2% Casamino acids (Acros, AC61204), 2 mM MgSO 4 (Sigma-Aldrich, 230391), 0.1 mM CaCl 2 (Sigma-Aldrich, 449709), and 0.4% D-glucose (Sigma-Aldrich, G8270). Antibiotics used to select for circuit plasmids were 100 mg/ml carbenicillin (Corning, 46-100), 50 mg/ml kanamycin (Sigma-Aldrich, K4000), and 50 mg/ml spectinomycin (Sigma-Aldrich, S9007). The inputs used for the sensor promoters were isopropyl b-D-1-thiogalactopyranoside (IPTG; Sigma-Aldrich, I6758), anhydrotetracycline hydrochloride (aTc; Sigma-Aldrich, 37919), L-arabinose (L-ara; Sigma-Aldrich, A3256), and N-(3-oxohexanoyl)-L-homoserine lactone (3OC6-HSL; Sigma-Aldrich, K3007).
8
Bacterial Strain Cultivation and Induction
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Bacterial strains were grown in Luria-Bertani (LB) (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) (Fisher Scientific, Waltham, MA, USA) liquid media or on agar plates (media with 1.5% agar) for plasmid construction and strain maintenance. All of the induction studies were done using M9 minimal media supplemented with 6.8 g/L Na 2 PO 4 , 3 g/L KH 2 PO 4 , 0.5 g/L NaCl, 1 g/L NH 4 Cl (all from Sigma-Aldrich, St. Louis, MO, USA), 2 mM MgSO 4 (Fischer Scientific), 100 µM CaCl 2 (Sigma-Aldrich), 0.4% glucose (Sigma-Aldrich), and 0.2% casamino acids (Acros Organics, Geel, Belgium). Both the LB and M9 media were supplemented with 20 µg/mL of chloramphenicol (Acros Organics), 25 µg/mL ampicillin (Acros Organics), and appropriate concentrations of isopropyl-D-1-thiogalactopyranoside (IPTG) (USB Molecular Biology Reagents/Affymetrix, Santa Clara, CA, USA), or L (+)-arabinose (Sigma-Aldrich). Phosphate-buffered saline (PBS) with 2 mg/mL kanamycin was utilized throughout the studies.
9
Anaerobic E. coli Cultivation and Supplementation
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E. coli K-12
BW25113 was acquired from VWR (470179-082). E. coli ΔmoaA was constructed as previously described.61 (link),62 (link) Archived stocks of bacteria were maintained in 16% glycerol at -80
°C. Bacteria were routinely cultured in modified M9 mineral media
(mM9, defined below) at 37 °C under anoxic and reducing conditions
(2–5% H2, 20% CO2, with the balance being
N2) in a COY anaerobic chamber. When indicated, mM9 media
(described below) was supplemented with 50 mM sodium nitrate (Sigma,
S5506-250G), 50 mM sodium nitrite (Fisher Scientific, M1065490100),
500 μM dopamine HCl (Alfa Aesar, A11136-06), and/or 20 μM
α-syn (purified as described below). Prior to beginning experiments,
the medium was equilibrated under anoxic conditions overnight to remove
oxygen.
mM9 medium was prepared to contain the following: 1x
M9 salts (Sigma, M6030-1KG), 2 mM magnesium sulfate (Fisher Scientific,
0338-500G), 100 μM calcium chloride (Fisher Scientific, AC219171000),
20 mMd -glucose anhydrous (VWR Life Science, Biotechnology
grade), 0.2% (w/v) casamino acids
(Fisher Scientific, DF0288-15-6), 1× vitamin supplement (ATCC,
MD-VS), and 500 μM ferrous iron chloride (Oakwood, 098678-5g).
BW25113 was acquired from VWR (470179-082). E. coli ΔmoaA was constructed as previously described.61 (link),62 (link) Archived stocks of bacteria were maintained in 16% glycerol at -80
°C. Bacteria were routinely cultured in modified M9 mineral media
(mM9, defined below) at 37 °C under anoxic and reducing conditions
(2–5% H2, 20% CO2, with the balance being
N2) in a COY anaerobic chamber. When indicated, mM9 media
(described below) was supplemented with 50 mM sodium nitrate (Sigma,
S5506-250G), 50 mM sodium nitrite (Fisher Scientific, M1065490100),
500 μM dopamine HCl (Alfa Aesar, A11136-06), and/or 20 μM
α-syn (purified as described below). Prior to beginning experiments,
the medium was equilibrated under anoxic conditions overnight to remove
oxygen.
mM9 medium was prepared to contain the following: 1x
M9 salts (Sigma, M6030-1KG), 2 mM magnesium sulfate (Fisher Scientific,
0338-500G), 100 μM calcium chloride (Fisher Scientific, AC219171000),
20 mM
grade), 0.2% (w/v) casamino acids
(Fisher Scientific, DF0288-15-6), 1× vitamin supplement (ATCC,
MD-VS), and 500 μM ferrous iron chloride (Oakwood, 098678-5g).
10
Reagents for Antimicrobial Susceptibility Testing
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Gentamicin, agarose, chloroform and casamino acids were obtained from Fisher Scientific (Ottawa, ON, Canada). Erythromycin was purchased from Caledon Laboratories LTD (Georgetown, WA, Canada). Phenylalanine-Arginine-β-Naphthylamide, cholesterol and Triton X-100 were purchased from Sigma Aldrich (Oakville, ON, Canada). The compound 2-Nitrophenyl-β-d -galactopyranoside was obtained from Thermo Fisher Scientific (Ottawa, ON, Canada). DPPC (1,2-Dipalmitoyl-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Mueller–Hinton broth (MHB) and Lysogeny broth (LB) and agar were purchased from Becton Dickinson (Francklin Lakes, NJ, USA) and Becton Dickinson Microbiology Systems (Oakville, ON, Canada), respectively. ABt medium and Z buffer were prepared as described previously [47 (link),51 (link)].
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