We used the strain P. putida KT2440 FliCS267C. In a previous study, the flagellin protein FliC was genetically modified by exchanging serine 267 with a cysteine in order to fluorescently label the flagella. It was shown that this exchange does not influence motility (19 (link)). We refer to this strain as the wild type. P. putida was grown in shaking culture at 30°C, and the required E. coli strains for cloning were grown at 37°C. For cloning and the swimming agar assay, cells were inoculated in lysogeny broth. For flagellar staining experiments, cells were grown overnight in tryptone broth (10 g/L tryptone [Applichem], 5 g/L NaCl).
Tryptone
Manufactured by AppliChem
Sourced in Germany
Tryptone is a type of enzymatic peptone derived from casein. It is commonly used as a nutrient source in microbiology and cell culture applications.
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Lab products found in correlation
Sodium hydroxide (naoh), by Merck Group (1 mentions)
Sodium chloride (nacl), by AppliChem (1 mentions)
Peptone, by Avantor (1 mentions)
Yeast extract, by Avantor (1 mentions)
Mgso4 7h2o, by Avantor (1 mentions)
Nh4 2so4, by Avantor (1 mentions)
R2a agar, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Yeast extract, by AppliChem (1 mentions)
Lb medium, by AppliChem (1 mentions)
Sodium azide nan3, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Tmb 3 3 5 5 tetramethylbenzidine, by Merck Group (1 mentions)
Glycerol, by AppliChem (1 mentions)
Bm chemiluminescence western blotting kit, by Roche (1 mentions)
Nickel sepharose 6 fast flow, by GE Healthcare (1 mentions)
Gel purification and plasmid mini extraction kits, by Bioneer (1 mentions)
Goat anti mouse igg antibody hrp conjugate, by Santa Cruz Biotechnology (1 mentions)
8 protocols using tryptone
1
Flagellar Labeling in Pseudomonas putida KT2440
2
Thermophilic Cellulolytic B. coagulans Cultivation
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All the experiments were carried out using B. coagulans MA-13 [24 (link)], a thermophilic and cellulolytic strain previously isolated and cultivated using a medium containing a glycine-buffered Brock’s basal salt solution, which is suitable for the cultivation of thermoacidophilic microorganisms [34 (link)–36 (link)].
LB medium was used as inoculation medium and contained 1% (w/v) tryptone (AppliChem), 1% (w/v) NaCl (AppliChem), 0.5% (w/v) yeast extract (VWR).
The seed medium contained final concentrations of 5% (v/v) molasses, 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR), 0.75% (w/v) (NH4)2SO4 (VWR), 0.35% (w/v) KH2PO4 (VWR), 0.07% (w/v) MgSO4·7H2O (VWR), and 1× trace metals and 1× vitamins, prepared according to [37 (link)]. To test the pre-adaptation of seed cultures, hydrolysate was added at final concentrations of 30%, 40%, 50%, 70% and 95% (v/v) to the seed medium.
The SSF medium was composed of 10% weight/weight (w/w) water insoluble solids (WIS) supplemented with 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR) and 0.05% (w/v) (NH4)2HPO4 (VWR). With this setup, the approximate concentrations of the microbial growth inhibitors acetic acid, furfural and 5-hydroxymethyl furfural were 0.58 g/L, 0.61 g/L, and 0.21 g/L, respectively. All media were adjusted to pH 5.5 by titration with 3 M NaOH (Merck).
LB medium was used as inoculation medium and contained 1% (w/v) tryptone (AppliChem), 1% (w/v) NaCl (AppliChem), 0.5% (w/v) yeast extract (VWR).
The seed medium contained final concentrations of 5% (v/v) molasses, 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR), 0.75% (w/v) (NH4)2SO4 (VWR), 0.35% (w/v) KH2PO4 (VWR), 0.07% (w/v) MgSO4·7H2O (VWR), and 1× trace metals and 1× vitamins, prepared according to [37 (link)]. To test the pre-adaptation of seed cultures, hydrolysate was added at final concentrations of 30%, 40%, 50%, 70% and 95% (v/v) to the seed medium.
The SSF medium was composed of 10% weight/weight (w/w) water insoluble solids (WIS) supplemented with 1% (w/v) yeast extract (VWR), 1% (w/v) peptone (VWR) and 0.05% (w/v) (NH4)2HPO4 (VWR). With this setup, the approximate concentrations of the microbial growth inhibitors acetic acid, furfural and 5-hydroxymethyl furfural were 0.58 g/L, 0.61 g/L, and 0.21 g/L, respectively. All media were adjusted to pH 5.5 by titration with 3 M NaOH (Merck).
3
Highly Motile P. putida KT2440 Culture
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4
Bacterial Swimming Assay Protocol
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Swimming assays were performed as described previously (79 (link)) with small modifications. In brief, 2.5 μL of E. coli overnight culture was transferred on a plate containing 10 g/L tryptone, 5 g/L NaCl, and 0.3% agar (AppliChem) and incubated at 37°C. After 7 to 15 h, the swimming area was measured, and the relative swimming distance was calculated using E. coli wild type as a reference.
5
Bacterial Growth Nutrient Requirements
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In order to assess nutrient requirements, bacteria grown in liquid Grace’s medium with 10% FBS were seeded onto R2A agar (Sigma) or onto agarified Grace’s medium containing inorganic salts, vitamins and carbon sources (sucrose, glucose and fructose), as well as one of two different nitrogen sources: (i) peptones from casein (Serva) and tryptone (AppliChem) 5 g/L each, or (ii) ammonium chloride 1 g/L. Bacteria were incubated in 24-well plates as described above.
6
Antimicrobial Activity of Gentamicin Eluate
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The antimicrobial activity of the eluted released gentamicin was determined using an agar diffusion test regime with a Gram-positive bacterium, Staphylococcus aureus, clinically isolated, as described before [13 (link)]. Bacteria were grown in a LB medium (2 g yeast extract, 4 g tryptone (both Applichem GmbH Darmstadt, Germany, 2 g NaCl (Sigma-Aldrich, Darmstadt, Germany) and 400 mL H2O double dest)) and plated on LB agar plates (LB medium containing 1.5% (w/v) agar (Applichem GmbH, Darmstadt, Germany)) to get a bacterial lawn. Fifty µL of the eluate were pipetted on filter discs (diameter = 12 mm), which were placed in the middle of the agar gel. After 24 h of cultivation at 37 °C, the extent of the inhibition zones was measured.
7
Fluorescent flagella labeling of engineered P. putida
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P. putida KT2440 FliCS267C (P. putida FliC) is a genetically modified strain from the wild-type P. putida KT2440 (31 (link)). The genetic modification causes a single cysteine substitution in the flagellar filament protein as described in (7 (link)). P. putida FliC was grown in 50 ml of tryptone broth [tryptone (10 g liter−1; AppliChem) and NaCl (5 g liter−1)] overnight, being shaken at 300 rpm and 30°C. The F-L staining of flagella with Alexa Fluor 488 followed the protocol of (7 (link)). Both the chemotaxis and the bulk experiments were performed in a μ-Slide Chemotaxis three-dimensional device (ibidi; see fig. S1A). The overnight cell culture that contained the highly motile bacteria was directly used for further experiments (cf. the Supplementary Materials for additional technical details).
8
Antibody Library Screening Protocol
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All of the chemicals used in this work were of biological grade. Tryptone, agar, and glycerol were obtained from Applichem (Darmstadt, Germany). NaCl and polyethylene glycol (PEG) 6000 were purchased from Scharlau (Barcelona, Spain). Primers used in this work were supplied from Bioron (Ludwigshafen, Germany) ordered via FAZA Biotech (Tehran, Iran). Gel purification and plasmid mini extraction kits were obtained from Bioneer (Daejeon, South Korea). A domain antibody (dAb) library kit was purchased from MRC HGMP Resource Centre. Anti M13horseradish peroxidase (HRP)-conjugated monoclonal antibody was prepared from Sino Biological (Beijing, China). TMB (3,3′,5,5′-tetramethylbenzidine) was obtained from Sigma-Aldrich (St. Louis, MO). agarose was from Invitrogen (Paisley, UK). Sodium azide (NaN 3 ) and methanol were from Merck (Darmstadt, Germany). A PCR master kit was purchased from CinnaGen (Tehran, Iran). Nickel sepharose 6 fast flow was prepared from GE Healthcare Life Sciences (Sweden). FGF-7 antibody (A-9) sc-515014 and goat anti-mouse IgG antibody HRP conjugate were from Santa Cruz Biotechnology, and anti-His antibody was from GE Healthcare/Sigma-Aldrich. The BM Chemiluminescence Western blotting kit was obtained from Roche (Berlin, Germany). The MCF-7 cell line was purchased from the Pasteur Institute of Iran.
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