Wild type and mutant pETM442 IKKγ (40–354) and pETM6T1-vFLIP (1–178) were transformed into BL21(DE3)Star (Invitrogen) that harbored the pRARE2 plasmid encoding rare tRNAs (Novagen). pET151 IKKβ (644–756) was transformed into Rosetta-gami B (DE3) (Novagen). The cells were grown in 100 ml of 2YT medium (1.6% (w/v) bacto-tryptone (Invitrogen), 1% bacto-yeast extract (Invitrogen) and 0.5% NaCl, adjusted to pH 7.2) containing chloramphenicol (35 μg ml−1) and supplemented with either ampicillin (100 μg ml−1) for IKKγ, kanamycin (24 μg ml−1) for vFLIP, or kanamycin (24 μg ml−1), ampicillin (100 μg ml−1) and tetracyclin (12.5 μg ml−1) for IKKβ, and grown at 37 °C overnight. 1 in 100 dilutions of overnight culture were inoculated into 2YT medium and grown to an OD600 of 1.0 at 37 °C before induction with 1 mm isopropyl 1-thio-β-d -galactopyranoside (IPTG) for IKKγ and vFLIP or 0.1 mm IPTG for IKKβ, and allowed to grow at: 30 °C for 3 h (IKKγ); 16 °C overnight (vFLIP); or 20 °C overnight (IKKβ). The cells were then harvested by centrifugation, washed with buffer A (200 mm NaCl, 25 mm Tris, pH 8.5) and stored at −70 °C.
Bacto yeast extract
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Bacto Yeast Extract is a nutritional supplement used in microbiology and cell culture applications. It provides essential nutrients, vitamins, and minerals to support the growth and proliferation of microorganisms and cells in laboratory environments.
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Lab products found in correlation
Bacto tryptone, by Thermo Fisher Scientific (6 mentions)
Bacto peptone, by Thermo Fisher Scientific (5 mentions)
Peptone, by Thermo Fisher Scientific (3 mentions)
Bacto casamino acids, by BD (3 mentions)
Horse serum, by Merck Group (2 mentions)
Columbia agar base ca, by Thermo Fisher Scientific (2 mentions)
Bl21 star de3, by Thermo Fisher Scientific (1 mentions)
Escherichia coli dh5α cells, by Thermo Fisher Scientific (1 mentions)
4 amino 5 methylamino 2 7 difluorofluorescein diacetate daf fm da, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
N naphthyl ethylenediamine, by Merck Group (1 mentions)
C myc antibody, by Merck Group (1 mentions)
L arginine, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride pmsf, by Roche (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Hepes free acid, by Research Products International (1 mentions)
E coli bl21 cells, by New England Biolabs (1 mentions)
Gmppnp, by Abcam (1 mentions)
L glutathione reduced, by Merck Group (1 mentions)
Ampicillin, by Research Products International (1 mentions)
30 protocols using bacto yeast extract
1
Bacterial Expression of Recombinant IKK Proteins
2
Purification and Characterization of Synechococcus Nitric Oxide Synthase
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L-arginine, tetrahydrobiopterin (BH4), sulfanilamide (SA), N-naphthyl-ethylenediamine (NED), c-Myc agarose column and c-Myc antibody were all purchased from Sigma-Aldrich. The NOS inhibitor L-NAME was obtained from Calbiochem (San Diego, CA). The polyclonal anti-SyNOS was acquired from Genscript (USA). Restriction enzymes, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF-FM DA), Bacto-yeast extract, IPTG and Escherichia coli DH5α cells were purchased from Invitrogen (USA). BL21 (DE3) pLys cells, and both pET24b and pET15b were obtained from Novagen (Madison, WI). Synechococcus PCC 7335 was purchased as part of the Pasteur Culture Collection (PCC, Paris, France).
3
Genetic Manipulation of Haloarchaea
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H. volcanii strains were cultured as previously described (Quax et al.,2018b ; Li et al., 2019 ). Genetic manipulation and gene expression based on the selection with uracil in ΔpyrE2 strains were performed with PEG 600 as described previously (Allers et al., 2004 ). The cells were cultured at 45°C or 42°C, under constant rotation at 120 rpm, in complete YPC medium containing 5% BactoTM yeast extract, 1% peptone (Oxoid, UK), 1% BactoTM Casamino acids (BD Biosciences, UK) or in selective CA medium containing only 5% BactoTM Casamino acids in 18% SW (Salt water, containing per liter 144 g NaCl, 21 g MgSO4 X 7H2O, 18 g MgCl2 X 6H2O, 4.2 g KCl, and 12 mM Tris HCl, pH 7.3). Plasmids based on pTA1228 (Brendel et al., 2014 ), with pyrE2 for selection with uracil, were constructed to express proteins in H. volcanii strains (Table S3 ). Plasmids based on pTA131 were used to create knock‐out constructs for the pop‐in pop‐out method based on the pyrE2 gene. Salt stable GFP and mCherry genes were introduced to pTA1228 plasmid, allowing the expression of N‐terminal and C‐terminal fluorescent fusion proteins in H. volcanii strains (Duggin et al., 2015 ).
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H. volcanii strains were cultured as previously described (Quax et al.,
4
Genetic Manipulation of Haloarchaea
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Growth and genetic manipulation of H. volcanii. H. volcanii strains were cultured as previously described (Quax et al., 2018b; Li et al., 2019) . Genetic manipulation and gene expression based on selection with uracil in ΔpyrE2 strains were performed with PEG 600 as described previously (Allers et al., 2004) . The cells were cultured shaking at 120 rpm at 45 °C or 42 °C in complete YPC medium containing 5% Bacto TM yeast extract, 1% peptone (Oxoid, UK), 1% BactoTM Casamino acids (BD Biosciences, UK) or in selective CA medium containing only 5% BactoTM Casamino acids in 18 % SW (Salt water, containing per liter 144 g NaCl, 21 g MgSO4 X 7H2O, 18 g MgCl2 X 6H2O, 4.2 g KCl, and 12 mM Tris HCl, pH 7.3). Plasmids based on pTA1228 (Brendel et al., 2014) , with pyrE2 for selection with uracil, were constructed to express proteins in H. volcanii strains (Table S3). Plasmids based on pTA131 were used to create knock-out constructs for the pop-in pop-out method based on the pyrE2 gene. Salt stable GFP and mCherry genes were introduced to pTA1228 plasmid, allowing expression of N-terminal and C-terminal fluorescent fusion proteins in H. volcanii strains (Duggin et al, Nature, 2015) .
5
Optimization of Elicitor Concentrations for Ginkgo Biloba Cell Culture
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A series of concentrations of the elicitor solutions were prepared. Chitosan (CH) (448877-50G, Sigma-Aldrich, St. Louis, MO, USA) (50, 100, 200, 300, 400, and 500 mg/L) was dissolved in 1% acetic acid water solution. Yeast extract (YE) (Bacto™ Yeast Extract, #212750, Gibco) (50, 100, 200, 300, 400, and 500 mg/L) was dissolved in water. The methyl jasmonate (MJ) (392707-5ML, Sigma-Aldrich) (0.01, 0.05, 0.1, 0.3, and 0.5 mM) was diluted with water, and salicylic acid (SA) (S7401-500G, Sigma-Aldrich) (0.01, 0.05, 0.1, 0.3, and 0.5 mM) was dissolved in water. All of the elicitor solutions were filter sterilized before being added to the suspension cell culture individually. The cells were collected and analyzed after incubation for 2 days. The optimal conditions of the elicitors were determined in terms of cell mass, cell viability, and GB yield.
6
Engineered Kluyveromyces marxianus Strain
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Kluyveromyces marxianus CBS 6556 ura3Δ his3Δ was used as a starting strain for all experiments described in this work. All constructed strains are listed in Table S2. Synthetic defined (SD) media was used for all plasmid-based expression experiments. The SD-U medium is defined as 6.7 g/L BD Difco™ Yeast Nitrogen Base without amino acids, 1.92 g/L Yeast Synthetic Drop-out Medium Supplements without uracil, and 20 g/L d -glucose. SD-H and SD-H-U are similar defined but with 0.75 g/L of CSM-His and CSM-His-Ura, respectively. For all pathway refactoring experiments and 2-PE biosynthesis analysis, K. marxianus strains were cultivated rich YPD medium (YPD: 10 g/L Gibco™ Bacto™ Yeast Extract, 20 g/L Gibco™ Bacto™ Peptone, 20 g/L d -glucose). 20 g/L agar was added to make solid agar plates. All yeast cultures were conducted in 250 mL baffled shake flasks containing 25 mL of appropriate media. Culturing was conducted in an INFORS HT Multitron incubation shaker with temperature control set to 30, 37 and 45 °C as needed.
7
Ras Protein Purification from E. coli
8
Cultivation of Salmonella Typhi Strain
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Salmonella enterica subsp. enterica sv. Typhi strain STH2370 (S. Typhi) was used as the parental strain (Valenzuela et al., 2014 (link)). The strain was routinely grown in liquid culture using Luria Bertani medium (Bacto tryptone [Gibco], 10 g/L; Bacto yeast extract [Gibco], 5 g/L; NaCl [Winkler], 5 g/L; prepared in distilled water) at 37°C with shaking. When required, the medium was supplemented with agar (15 g/L), ampicillin (Amp, AppliChem GmbH), polymyxin B sulfate (Pmb, AppliChem GmbH), or meropenem (Mer, Sigma Aldrich). For S. Typhi, 1× Amp represents a concentration equivalent to the Minimum Inhibitory Concentration (MIC) of Amp (6.25 μg/mL), while 1× Pmb corresponds to the MIC of polymyxin B (0.31 μg/mL). Various concentrations based on these standards were employed, such as 0.25× Amp (1.56 μg/mL), 0.5× Amp (3.13 μg/mL), 2× Pmb (0.63 μg/mL), and 4× Pmb (1.25 μg/mL).
9
Bacterial Strain Growth and Supplementation
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The bacterial strains, plasmids used in this work are listed in S1 Table . Single colonies of E. coli strains grown overnight on lysogeny broth (LB) (Bacto Tryptone (#211699, Gibco) 10g/L, Bacto Yeast Extract (#212750, Gibco) 5g/L, NaCl 5 g/L) [58 (link)] agar (1.5% w/v) plates were picked and grown overnight in LB at 37°C for all experiments. Other bacterial growth media used in this study were Mueller Hinton (#275730, BD), MacConkey Agar (#CM0115, Oxiod). Where appropriate, media were supplemented with ampicillin (Amp, 100 μg/ml), kanamycin (Kan, 50 μg/ml), chloramphenicol (Chl, 25 μg/ml), bile salts sodium deoxycholate (DOC, 0.1% w/v, #D6750, Sigma), vancomycin (Van, 100 μg/ml, #SBR00001, Sigma), L-arabinose (0.2% w/v), D-glucose (0.2% w/v), crystal violet (0.0005% w/v), and colicin E2 (20 μg/ml).
10
Candida Species Cultivation and Inhibitors
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Escherichia coli ME9806 (iVEC3) (National Bio-Resource Project (NBRP), Mishima, Japan) was used as the cloning host. Candida auris CBS 10913, C. tropicalis CBS 94, C. parapsilosis CBS 604, and C. krusei CBS 573 were obtained through the ME9806 National Bio-Resource Project (NBRP), Chiba, Japan. The bacterial strains were grown in Luria Broth containing 50 µg/mL ampicillin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The strains used in the present study are listed in Table 1 . The YEp352-GAPII (containing TDH3 promoter and URA3) and the YEp351-GAPII plasmid (containing TDH3 promoter and LEU2) [45 (link)] were used to express recombinant proteins. All yeast strains were grown in YPD medium composed of 1% (w/v) Bacto Yeast Extract (Gibco, Miami, FL, USA), 2% (w/v) HIPOLYPEPTON (Nihon Pharmaceutical Co., Ltd., Osaka, Japan), and 2% (w/v) glucose or synthetic defined minimal medium (SD) (0.17% (w/v) Yeast Nitrogen Base without amino acids and ammonium sulfate, 5% (w/v) ammonium sulfate (Wako), 2% (w/v) glucose, and appropriate amino acids). The solid media were supplemented with 2% (w/v) agar (Wako). PF1163B (kindly provided by Meiji Seika Pharma Co., Ltd., Odawara, Kanagawa, Japan) and 1181-0519(N-[(2E)-2-[(4-nitrophenyl) hydrazinylidene] propyl] acetamide) (ChemDiv, San Diego, CA, USA) were used as growth inhibitors.
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