The C. urealyticum DSM 7109 strain obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen was grown in 1000 ml baffled Erlenmeyer flasks containing 250 ml of brain-heart infusion (BHI) medium (Becton) and 250 ml Erlenmeyer flasks containing 25 ml BYT medium [34 (link)]. The Corynebacterium strains C. glutamicum ΔporHΔporA [30 (link)] and C. glutamicum ΔporHΔporA expressing cur_1714 were grown in BHI broth and were stirred on a rotary shaker at 200 rpm and 30 °C. Escherichia coli NEB5α (New England Biolabs) and E. coli BL21 DH5α (ThermoFisher Scientific) were used for cloning and grown under standard conditions in Luria broth (LB) at 37 °C, shaken at 250 rpm. Agar plates and liquid media were supplemented with 25 μg/ml chloramphenicol, respectively, if required. Porin deficient BL21 DE3 Omp8 E. coli strains [30 (link)] were used for GST tagging and expressing of cur_1714 and cur_1715, and were grown at 37 °C under standard conditions in Luria broth (LB) while shaking at 250 rpm. Agar plates and liquid media were supplemented with kanamycin (25 μg/ml) or ampicillin (100 μg/ml) respectively, if necessary.
Escherichia coli neb5α
Manufactured by New England Biolabs
Sourced in United States
Escherichia coli NEB5α is a laboratory strain of E. coli bacteria designed for DNA cloning and plasmid propagation. It is a highly competent bacterial strain suitable for high-efficiency transformation of plasmids.
Automatically generated - may contain errors
Lab products found in correlation
Bhi medium, by BD (1 mentions)
Cobalt chloride, by Merck Group (1 mentions)
Calcium sulphate, by Merck Group (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Magnesium sulphate, by Merck Group (1 mentions)
Ferrous sulphate, by Merck Group (1 mentions)
Zinc chloride, by Merck Group (1 mentions)
Agar, by Carl Roth (1 mentions)
Potassium dihydrogen phosphate, by Carl Roth (1 mentions)
Sodium iodide, by Merck Group (1 mentions)
Ammonium sulphate, by Carl Roth (1 mentions)
Potassium hydrogen phosphate, by Carl Roth (1 mentions)
Glucose, by Carl Roth (1 mentions)
Copper sulphate, by Merck Group (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Biotin, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
Ethanol, by Avantor (1 mentions)
Glycerol, by Carl Roth (1 mentions)
Zeocin, by Thermo Fisher Scientific (1 mentions)
15 protocols using escherichia coli neb5α
1
Cultivation of Corynebacterium and E. coli Strains
2
Clostridium difficile Strain Cultivation Protocol
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C. difficile 63035 (link) and a previously constructed ΔsrtB mutant19 (link) were grown under anaerobic conditions (80% N2, 10% CO2, 10% H2) in an anaerobic incubator (Don Whitley Scientific) at 37 °C on Brain Heart Infusion Supplemented (BHIS) plates or in broth [BHI supplemented with 5 g/l yeast extract (BD Bacto), and 1 g/l L-cysteine, and 15 g/l agar (Bacto) for plates]. Growth was supplemented with thiamphenicol (15 μg/ml) for plasmid propagation, cycloserine (250 μg/ml) and cefoxitin (25 μg/ml) for counter selection against E. coli during conjugation, and anhydrotetracycline (ATc 100 ng/ml) for induction of Ptet expression vectors36 (link). Escherichia coli NEB5α (New England Biolabs) was used for cloning and plasmid propagation, strain CA434 (HB101 carrying R702) was used for conjugation of plasmids into C. difficile, and Rosetta (Novagen, carrying pRARE) was used for expression of proteins for purification. E. coli strains were grown on LB agar and in LB broth supplemented with chloramphenicol (15 μg/ml) and kanamycin (50 μg/ml).
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C. difficile 63035 (link) and a previously constructed ΔsrtB mutant19 (link) were grown under anaerobic conditions (80% N2, 10% CO2, 10% H2) in an anaerobic incubator (Don Whitley Scientific) at 37 °C on Brain Heart Infusion Supplemented (BHIS) plates or in broth [BHI supplemented with 5 g/l yeast extract (BD Bacto), and 1 g/l L-cysteine, and 15 g/l agar (Bacto) for plates]. Growth was supplemented with thiamphenicol (15 μg/ml) for plasmid propagation, cycloserine (250 μg/ml) and cefoxitin (25 μg/ml) for counter selection against E. coli during conjugation, and anhydrotetracycline (ATc 100 ng/ml) for induction of Ptet expression vectors36 (link). Escherichia coli NEB5α (New England Biolabs) was used for cloning and plasmid propagation, strain CA434 (HB101 carrying R702) was used for conjugation of plasmids into C. difficile, and Rosetta (Novagen, carrying pRARE) was used for expression of proteins for purification. E. coli strains were grown on LB agar and in LB broth supplemented with chloramphenicol (15 μg/ml) and kanamycin (50 μg/ml).
3
Heterologous Expression of Enzymes in P. pastoris
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All chemicals used were of analytical grade or highest purity available. All buffers and aqueous solutions were prepared with deionised water (>17 MΩ). 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and yeast nitrogen base were from Amresco LLC (OH, USA). 2,6-dimethoxyphenol (DMP), acetic acid, boric acid, magnesium chloride, manganese sulphate, peptone from casein, potassium hydroxide, potassium sulphate, sodium hydrogen phosphate, sodium molybdate and sulfuric acid were from Fluka (St. Gallen, CH). Zeocin was from Invitrogen (Carlsbad, CA, USA). Sodium hydroxide was from Merck (Darmstadt, Germany). Agar, ammonium sulphate, glucose, glycerol, mEthanol, phosphoric acid (85%), potassium sulphate, potassium dihydrogen phosphate, potassium hydrogen phosphate and sodium chloride were from Roth (Karlsruhe, Germany). Agarose, biotin, calcium sulphate, citric acid, cobalt chloride, copper sulphate, EDTA, ferrous sulphate, magnesium sulphate, sodium iodide, Tris base, yeast extract and zinc chloride were from Sigma Aldrich (St. Louis, USA). Ethanol was from VWR (Radnor, USA). Escherichia coli NEB5α (New England Biolabs, Ipswich, MA, USA) was used for subcloning and Pichia pastoris × 33 (Invitrogen) for heterologous expression9 (link). A modified pGAPZ A vector (Invitrogen) under the control of the GAP promotor was used for expression in P. pastoris43 (link).
4
Bacterial Strain Characterization for recA Gene
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All chemicals were purchased from Sigma-Aldrich, Burlington, USA, if not stated otherwise. Bacterial strains used in this work were Escherichia coli K12 (Addgene, Watertown, MA, USA), Escherichia coli TOP10 (Invitrogen, Carlsbad, CA, USA), Escherichia coli NEB 5α (New England Biolabs, Ipswich, USA) and Staphylococcus carnosus (kindly provided by Dr. K. Plätzer, University of Salzburg). The respective strains were chosen based on the presence of the recA gene in their genome. A list stating which strains carry a functional copy can be seen in Table 1 .
5
Allelic Replacement of Listeria hlyA Gene
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The bacterial source strains used for this work were Escherichia coli NEB5α (New England BioLabs) for plasmid constructions and Listeria monocytogenes LL195 [17 ] for all of the experiments involving Lm. All strains were grown at 37°C under shaking at 190 rpm in Luria Bertani (LB) medium for E. coli, in brain heart infusion (BHI) for Lm. Whenever required, media were supplemented with antibiotics for plasmid selection (chloramphenicol, 35 μg/ml for E. coli; 7 μg/ml for Lm).
For allelic replacement at the hlyA locus, the pMAD-∆hlyA plasmid was created by amplifying two partially overlapping fragments by PCR: 1,000 base pairs (bp) upstream (plcA gene) and downstream (mpl gene) of the hlyA open reading frame in the LL195 genome were amplified, respectively, with oligonucleotides oAL647-50 and oAL648-9 (Table S6). These fragments were inserted into the pMAD vector between the SalI and BglII restriction sites by Gibson Assembly, using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Allelic replacement of the hlyA open reading frame by these constructs, in the genome of L. monocytogenes strain LL195, was obtained as previously described [45 (link),46 (link)].
For allelic replacement at the hlyA locus, the pMAD-∆hlyA plasmid was created by amplifying two partially overlapping fragments by PCR: 1,000 base pairs (bp) upstream (plcA gene) and downstream (mpl gene) of the hlyA open reading frame in the LL195 genome were amplified, respectively, with oligonucleotides oAL647-50 and oAL648-9 (Table S6). These fragments were inserted into the pMAD vector between the SalI and BglII restriction sites by Gibson Assembly, using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). Allelic replacement of the hlyA open reading frame by these constructs, in the genome of L. monocytogenes strain LL195, was obtained as previously described [45 (link),46 (link)].
6
Shuttle Vector Construction in E. coli and Z. mobilis
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All plasmid construction was performed using Escherichia coli NEB5α from NewEnglandBiolabs (NEB), grown in LB0 medium (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) at 37°C with shaking. Experiments for the characterization of shuttle vectors were performed with Z. mobilis strain ATCC 31821 (ZM4). Cultivations were performed at 30°C in complex medium (ZM: Bacto peptone 10 g/L, yeast extract 10 g/L, glucose 20 g/L). Kanamycin and chloramphenicol were used at 100 μg/mL, spectinomycin was used at 150 μg/mL. Bacto agar 15 g/L was added for solid media.
7
Genetic manipulation of Lactobacillus plantarum
8
Bacterial Cultivation and Transformation
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Escherichia coli NEB5α (New England BioLabs) and BL21(DE3) (Novagen) were grown in LB broth, Miller (Difco), with aeration at 250 rpm. Plasmids were transformed into cells by electroporation, and transformants were selected by plating on LB agar with the appropriate antibiotic and were incubated at 37°C overnight. For work in M. smegmatis , MC2155 was used (K. Derbyshire) and the conditions employed are described below.
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9
Yeast Strain and Plasmid Management
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S. cerevisiae strains and plasmids used in this study are listed in Table 1 . Escherichia coli NEB 5α (New England BioLabs, Ipswich, MA, USA) was used for subcloning. All strains were stored in 20 % glycerol at −80 °C. Yeast strains were grown on solid YNB media (6.7 g/l yeast nitrogen base without amino acids, 20 g/l glucose and 20 g/l agar) before they were inoculated in the preculture as described below.
10
Engineered Lactiplantibacillus plantarum Amylase Expression
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Lactiplantibacillus plantarum WCSF1, isolated from human saliva as described by Kleerebezem et al. (2003) (link), was originally obtained from NIZO Food Research (Ede, Netherlands) and maintained in the culture collection of the Norwegian University of Life Sciences (NMBU), Ås, Norway. L. plantarum WCSF1 was used as an expression host for the vectors harboring α-amylase genes. L. plantarum strains were grown anaerobically in deMan, Rogosa, and Sharpe (MRS) broth (Carl Roth, Karlsruhe, Germany) at 37°C without agitation. All solid media were prepared with the supplementation of 1.5% (w/v) agar. Escherichia coli NEB5α (New England Biolab, Frankfurt am Main, Germany) was used in the transformation involving subcloning of DNA fragments and grown in Luria-Bertani (LB) broth at 37°C with agitation at 180 rpm. To maintain the plasmids, erythromycin was added into the cultivation media to final concentrations of 200 μg/mL for E. coli and 5 μg/mL for L. plantarum.
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