Bacterial strains, reagents, and growth conditions M. tuberculosis H37Rv, Mycobacterium smegmatis (Mc2 155), and Escherichia coli XL1 blue (Agilent Technologies, Santa Clara, CA) were used. M. tuberculosis were grown in Middlebrook 7H9 broth (liquid) and Middlebrook 7H10 agar (solid) (Difco, Franklin Lakes, NJ), supplemented with 0.05% Tween 80, 0.2% glycerol, and 10% ADN (2% glucose, 5% bovine serum albumin, 0.15 M NaCl). However, 10% AND was excluded from the 7H9 or 7H10 media while M. smegmatis were grown. For DNA cloning, Escherichia coli XL1 blue (Agilent Technologies, Santa Clara, CA) were grown in Luria-Bertani (LB) broth or agar (Thermo Fisher Scientific, Waltham, MA). As needed, solid and liquid media were supplemented with 25 or 50 µg ml -1 kanamycin sulfate (Thermo Fisher Scientific, Waltham, MA) for Mycobacterium and E. coli, respectively. M. smegmatis knock outs in mprA (hygromycin marked) and sigE (zeomycin marked) were obtained from
Escherichia coli xl 1 blue
Manufactured by Agilent Technologies
Sourced in United States
Escherichia coli XL-1 blue is a competent bacterial strain used in molecular biology applications. It is a genetically engineered strain of the common bacterium Escherichia coli, designed for increased transformation efficiency and improved plasmid stability.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Oligonucleotide, by Thermo Fisher Scientific (1 mentions)
Quikchange mutagenesis kit, by Agilent Technologies (1 mentions)
Spectinomycin, by Merck Group (1 mentions)
Apramycin, by Merck Group (1 mentions)
Phosphomycin, by Merck Group (1 mentions)
Carbenicillin, by Merck Group (1 mentions)
Moclo toolkit, by Addgene (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Kanamycin sulfate, by Thermo Fisher Scientific (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Mannitol salt agar, by Nissui Pharmaceutical (1 mentions)
Mueller hinton 2 broth, by BD (1 mentions)
Cetrimide agar, by Nissui Pharmaceutical (1 mentions)
Lb broth, by Nacalai Tesque (1 mentions)
9 protocols using escherichia coli xl 1 blue
1
Mycobacterial Growth and Genetic Manipulation
2
Enzymatic Reduction of Aldehydes
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Buffer components and other bulk chemicals were of the highest purity commercially available. Propanal (1 , ≥ 97%) (Sigma/Aldrich #W292303, Darmstadt, Germany) and (S)‐lactaldehyde (2 , 1 m solution in H2O) (Sigma/Aldrich #47014), NADH (disodium salt, Sigma/Aldrich #N8129) and NAD+ (Sigma/Aldrich #443410) were used in reactions as provided by the manufacturer. Oligonucleotides were custom synthesized by Thermo Scientific, Heidelberg, Germany. QuikChange mutagenesis kit and Escherichia coli XL‐1 Blue were purchased from Agilent, Santa Clara, CA, USA.
3
Isolation and Characterization of Streptomyces
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The isolation and phylogenetic characterization of Streptomyces sp. IB2014/011-12 were reported in (Axenov-Gribanov et al., 2016 (link)). Streptomyces strains were grown on solid nutrient medium MS (mannitol soy flour agar) and in liquid TSB medium (Kieser, 2000 ). For secondary metabolite production, NL19 (MS medium without agar) and SG (glucose, yeast, Bacto Soytone, and calcium carbonate) medium have been used. Escherichia coli XL1Blue (Agilent, United States) was used for routine cloning, and E. coli MW 6026 was used as a donor in the intergenic conjugation (Blodgett et al., 2007 (link)). E. coli strains were grown in Luria-Bertani (LB) broth. For MW 6026, diaminopimelic acid was added. When required, antibiotics were added to the cultures at the following concentrations: 50 μg ml−1 apramycin, 100 μg ml−1 spectinomycin, 100 μg ml−1 phosphomycin, and 100 μg ml−1 carbenicillin (Sigma, United States; Roth, Germany).
4
Genetic Construction of AMPs
5
Recombinant Production and Purification of ELdcRs
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All ELdcRs were recombinantly produced as described elsewhere. 29 (link) Briefly, the ELdcR-encoding genes were constructed into a pDrive vector in Escherichia coli XL-1 blue (Agilent, USA) then cloned into a pET-25b (+) vector for expression in E coli BLR (DE3) strain. After bacterial fermentation in a 15-L bioreactor (Applikon biotechnology, USA), the ELdcRs were purified by inverse transition cycling (ITC) using 1.5 M NaCl for precipitation. Pure products were dialyzed against deionized and ultrapure water The theoretical hydrophobicity of the ELdcRs was calculated using the ProtScale algorithm and the Kyte-Doolittle scale. 30, (link)31 (link) The algorithm predicts the hydrophilic and hydrophobic tendencies of a polypeptide chain by the progressive evaluation (from the N-terminus to the C-terminus) of the average hydropathy following the Kyte-Doolittle scale, where the larger the number is, the more hydrophobic the amino acid. The most hydrophilic amino acids are arginine (-4.5) and lysine (-3.9), whereas the most hydrophobic ones are isoleucine (4.5) and valine (4.2).
6
Modular Genetic Assembly in E. coli
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Native genetic parts were amplified from plasmids using Q5 High-Fidelity DNA Polymerase (New England Biolabs). Where necessary, native genetic parts were made compatible, (i.e., BsaI and BpiI sites were removed) using specific primers. Golden Gate assembly reactions were performed with restriction enzymes BsaI (Thermo Fisher Scientific) or BpiI (Thermo Fisher Scientific), and T4 DNAligase (Thermo Fisher Scientific) according to the protocol of the MoClo Toolkit (Addgene kit # 1000000044). Vectors were transformed into chemically competent Escherichia coli XL-1 blue (Agilent) as per the manufacturer’s instructions. Transformed cultures were grown at 37°C on LB medium with appropriate antibiotic selection for levels 0 and 1 vectors from the MoClo Toolkit (Addgene kit # 1000000044) which are respectively destination vectors for single genetic element and assembled transcription unit as outlined in (Weber et al., 2011 (link)). Four final vectors were constructed where uidA and NAT genes, separated by a 2A peptide, are under control of the Phaeodactylum pFcpB promoter/pFcpA terminator.
7
Cultivation of E. coli and Thermus thermophilus
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Escherichia coli XL-1 Blue (Agilent Technologies, Santa Clara, USA) was used as a host for DNA manipulations and was grown in LB medium (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) at 37°C. T. thermophilus HB27 (DSM 7039) and its derivative strains were grown at 60°C or 70°C with vigorous shaking in rich medium (TB) or nutritionally defined medium (SH). TB medium had a pH of 7.5 and contained per litre 8 g trypticase peptone, 4 g yeast extract, and 3 g NaCl, and was prepared with a high-carbonate mineral water (Purania, DRINKPOOL GmbH, Germany). SH medium was prepared as described in [60 (link)]. The growth media were supplemented with ampicillin (100 μg/ml for E. coli), kanamycin (20 μg/ml for E. coli and T. thermophilus), bleomycin (“Bleocin”, Calbiochem, 15 μg/ml), chloramphenicol (12.5 μg/ml for E. coli), XGlc (5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 50 μg/ml) or XGal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 50 μg/ml) when appropriate. All reagents were purchased from Sigma-Aldrich (Schnelldorf, Germany) except for growth media components which were obtained from BD Biosciences (Heidelberg, Germany).
8
Microbial Strain Acquisition and Molecular Cloning
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All of the chemicals were purchased from Tokyo Chemical Industry Co. Ltd, Sigma-Aldrich and Wako Pure Chemical Industries Ltd unless otherwise specified. Purchased chemicals were of reagent grade and used without further purification. M. robertsii ARSEF 23 was obtained from the United States Department of Agriculture-Agricultural Research Service. A. nidulans A1145 was obtained from the Fungal Genetics Stock Center in the USA. Escherichia coli XL1-Blue (Agilent Technologies, Santa Clara, CA, USA) and DNA restriction enzymes were used as recommended by the manufacturer (Thermo Fisher Scientific Inc., Waltham, MA, USA). Polymerase chain reaction was carried out using PrimeSTAR GXL DNA polymerase (TAKARA Bio Inc., Kusatsu City, Japan) as recommended by the manufacturer. Sequences of polymerase chain reaction products were confirmed through DNA sequencing (Macrogen Japan Corporation, Kyoto City, Japan). Saccharomyces cerevisiae BY4741 27 used for homologous recombination-based molecular cloning of genes and plasmid assembly was obtained from the Yeast Genetic Resource Center in Japan.
9
Antimicrobial Activity of Fly Larvae
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Musca domestica larvae were provided by E's Inc. (Tokyo, Japan). Staphylococcus aureus (NBRC100910), Staphylococcus epidermidis (NBRC100911), and Pseudomonas aeruginosa (NBRC12689) were purchased from National Institute of Technology and Evaluation (Kisarazu, Chiba, Japan). Escherichia coli XL1-blue was purchased from Stratagene (Agilent Technologies, Santa Clara, California, USA). Mannitol salt agar and cetrimide agar were purchased from Nissui Pharmaceutical (Tokyo, Japan). Mueller-Hinton II broth was purchased from Becton Dickinson (Franklin Lakes, New Jersey, USA). LB broth was purchased from Nacalai tesque (Kyoto, Japan). Agar for bacterial culture was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
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