Spectinomycin

Manufactured by GoldBio

Spectinomycin is a broad-spectrum antibiotic used in laboratory settings. It functions as an inhibitor of bacterial protein synthesis, preventing the growth and replication of targeted microorganisms.

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Lab products found in correlation

Kanamycin, by GoldBio (9 mentions) Ampicillin, by GoldBio (5 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (2 mentions) Bovine serum albumin standard, by Thermo Fisher Scientific (2 mentions) Thiamine hydrochloride, by Merck Group (2 mentions) Casamino acid, by Thermo Fisher Scientific (2 mentions) D glucose, by Merck Group (2 mentions) Cacl2, by Merck Group (2 mentions) Mgso4, by Merck Group (2 mentions) Citrulline, by Merck Group (1 mentions) Arginine, by Merck Group (1 mentions) Ornithine, by Merck Group (1 mentions) 0.22 μm filter, by Merck Group (1 mentions) Lb agar, by BD (1 mentions) L arabinose ara, by Merck Group (1 mentions) Anhydrotetracycline hydrochloride atc, by Merck Group (1 mentions) Isopropyl β d 1 thiogalactopyranoside iptg, by Merck Group (1 mentions) Neb 10 beta, by New England Biolabs (1 mentions) Anhydrotetracycline hydrochloride, by Merck Group (1 mentions) Neb 5 alpha, by New England Biolabs (1 mentions)

12 protocols using spectinomycin

Genetic Manipulation of UPEC Strains

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The UPEC strains used in this study are all derivatives of the human cystitis isolate UTI89 and are listed in Table S1 (17 (link), 91 (link)). In general, bacteria were grown and propagated in Luria-Bertani (LB) broth and plated for isolation on LB agar (BD). Where necessary, the medium was supplemented with 50 μg/ml kanamycin, 50 μg/ml spectinomycin, 100 μg/ml ampicillin, and/or 1 mM isopropyl β-d-1-thiogalactopyranoside (Gold-Bio). Human urine was collected from at least 2 healthy volunteers and filter sterilized through a 0.22-μm filter (Millipore). Biological replicates of growth curves were conducted with different batches of urine collected on different days. Where indicated, urine was supplemented with 10 mM ornithine, citrulline, or arginine (Sigma-Aldrich).
All deletion mutants were generated in the prototypical cystitis isolate UTI89 using the λRed recombinase system (92 (link), 93 (link)). The allelic replacement of argI was performed using a previously described, λRed recombinase-based negative-selection system (65 (link)). To facilitate in vivo competition assays, kanamycin or chloramphenicol resistance markers were inserted into the HK site using the standard λRed recombinase protocol (49 (link), 94 (link)). All deletion and allelic replacement mutants were confirmed by Sanger sequencing.

Hibbing M.E., Dodson K.W., Kalas V., Chen S.L, & Hultgren S.J. (2020). Adaptation of Arginine Synthesis among Uropathogenic Branches of the Escherichia coli Phylogeny Reveals Adjustment to the Urinary Tract Habitat. mBio, 11(5), e02318-20.

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Transposon Mutagenesis in Invasive GAS

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Bacterial strains used in this study are listed in S4 Table. GAS 5448 is a representative of the globally disseminated invasive serotype M1T1 clone isolated from a patient with necrotizing fasciitis and toxic shock [66 (link)]. The Krmit mutant library in GAS 5448 was produced by in vivo mariner mutagenesis [43 (link)] and contains over 85,000 independent mariner transposants (i.e., one Krmit TIS for every 22 nucleotides). GAS strains were routinely cultured in Todd-Hewitt medium (Alpha Biosciences) supplemented with 0.2% yeast extract (THY) as described [97 ]. Escherichia coli strain DH5α was routinely used as host for plasmid construction and preparation and cultured in Luria-Bertani (LB) medium (EMD Chemicals). Antibiotics (Gold Biotechnology) were used at the following concentrations: Spectinomycin (Sp) at 100 μg/ml for both E. coli and GAS, and Kanamycin (Km) at 50 μg/ml for E. coli and 300 μg/ml for GAS.

Le Breton Y., Belew A.T., Freiberg J.A., Sundar G.S., Islam E., Lieberman J., Shirtliff M.E., Tettelin H., El-Sayed N.M, & McIver K.S. (2017). Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection. PLoS Pathogens, 13(8), e1006584.

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Purification and Characterization of Flavodoxin and Reductase

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Ferric chloride was obtained from EMD Biosciences (Gibbstown, NJ). Bradford reagent for protein concentration determination as well as the bovine serum albumin (BSA) standard were purchased from Pierce, Thermo Fisher Scientific (Rockford, IL). Dithiothreitol (DTT), L-(+)-arabinose, isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, spectinomycin, and kanamycin were purchased from Gold Biotechnology (St. Louis, MO). SUMO express protease was purchased from Lucigen (Middleton, WI). SAM was synthesized from ATP and L-methionine and purified as described previously.^{10 (link)}E. coli flavodoxin (flv) and flavodoxin reductase (flr) were purified as described previously.^{11 (link)} Nick columns containing Sephadex G-50 resin were obtained from GE Healthcare (Buckinghamshire, UK). All reagents for chemical syntheses were obtained from Sigma–Aldrich unless specified further. All other materials and reagents have been previously reported,^{3 (link), 4 (link)} or were of the highest available quality.

Blaszczyk A.J., Knox H.L, & Booker S.J. (2019). Understanding the Role of Electron Donors in the Reaction Catalyzed by TsrM, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 24(6), 831-839.

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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).

Espah Borujeni A., Zhang J., Doosthosseini H., Nielsen A.A, & Voigt C.A. (2020). Genetic circuit characterization by inferring RNA polymerase movement and ribosome usage. Nature Communications, 11, 5001.

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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.

Lebovich M., Zeng M, & Andrews L.B. (2023). Algorithmic Programming of Sequential Logic and Genetic Circuits for Recording Biochemical Concentration in a Probiotic Bacterium. ACS Synthetic Biology, 12(9), 2632-2649.

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GAS Bacterial Strain Cultivation and Antibiotic Usage

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Bacterial strains used in this study are shown in Table S8. 544872 (link) is a GAS clinical isolate representative of the globally disseminated invasive serotype M1T1 clone. NZ13173 (link) is a GAS strain isolated from a patient with Acute Post-Streptococcal Glomerulonephritis (APSGN). GAS strains were routinely cultured in Todd-Hewitt medium (Alpha Biosciences) supplemented with 0.2% yeast extract (THY) as described elsewhere74 (link). Escherichia coli strains DH5α75 (link) and C43[DE3]76 (link) were used as hosts for plasmid construction and preparation and were cultured in Luria-Bertani (LB) medium (EMD Chemicals). Antibiotics (Fischer Scientific; Gold Biotechnology) were used at the following concentrations: Ampicillin (Ap) at 100 μg/ml for E. coli, Spectinomycin (Sp) at 100 μg/ml for both E. coli and GAS, and Kanamycin (Km) at 50 μg/ml for E. coli and 300 μg/ml for GAS.

Le Breton Y., Belew A.T., Valdes K.M., Islam E., Curry P., Tettelin H., Shirtliff M.E., El-Sayed N.M, & McIver K.S. (2015). Essential Genes in the Core Genome of the Human Pathogen Streptococcus pyogenes. Scientific Reports, 5, 9838.

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Bacillus subtilis Transformation Protocol

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B. subtilis was inoculated into 1 mL MC medium (Loyo and Burton, 2018 ) and incubated for 4 hours at 37°C for transformation. Plasmid DNA was f irst linearized by treatment with ScaI restriction enzyme (New England Biolabs). Next, 200 ng of plasmid DNA was added to 200 ml cell culture and incubated for 2 hours at 37°C. Tr ansformed cells were selected by plating on LB plates containing 100 mg ml⁻¹ spectinomycin (Gold Biotechnology). Plasmid pVP038 (Table S10) was transformed into B. subtilis 168, trpC2, cat to make B. subtilis 168, trpC2, cat, amyE::Pveg-gfp-spec (Table S9).

Hsu R.H., Clark R.L., Tan J.W., Ahn J.C., Gupta S., Romero P.A, & Venturelli O.S. (2019). Microbial interaction network inference in microfluidic droplets. Cell systems, 9(3), 229-242.e4.

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Purification of Flavodoxin Proteins

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Ferric chloride was obtained from EMD Biosciences (Gibbstown, NJ). Bradford reagent for protein concentration determination as well as the bovine serum albumin (BSA) standard was purchased from Pierce, Thermo Fisher Scientific (Rockford, IL). Dithiothreitol (DTT), L-(+)-arabinose, isopropyl β-D-1-thiogalactopyranoside (IPTG), ampicillin, spectinomycin, and kanamycin were purchased from Gold Biotechnology (St. Louis, MO). SUMO express protease was purchased from Lucigen (Middleton, WI). SAM was synthesized from ATP and L-methionine and purified as described previously.^{19 (link)}E. coli flavodoxin (flv) and flavodoxin reductase (flr) were purified as described previously.^{20 (link)} All reagents for chemical syntheses were obtained from Sigma–Aldrich unless specified further. All other materials and reagents have been previously reported,^{21 (link), 22 (link)} or were of the highest available quality.

Wang B., Blaszczyk A., Knox H.L., Zhou S., Blaesi E.J., Krebs C., Wang R.X, & Booker S.J. (2018). Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase. Biochemistry, 57(33), 4972-4984.

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Engineered E. coli Strain for Plasmid Cloning

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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 for cloning and measurements (New England Biolabs, MA, C3019). Cells were grown in LB Miller broth (Difco, MI, 90003‐350) for harvesting plasmids. Cells were grown in MOPS EZ Rich (Teknova, M2105) defined medium with 0.2% glycerol for circuit performance measurements. To select for the presence of plasmids, 50 μg/ml kanamycin (Gold Biotechnology, MO, K‐120‐5) and 50 μg/ml spectinomycin (Gold Biotechnology, MO, S‐140‐5) antibiotics were used.

Gorochowski T.E., Espah Borujeni A., Park Y., Nielsen A.A., Zhang J., Der B.S., Gordon D.B, & Voigt C.A. (2017). Genetic circuit characterization and debugging using RNA‐seq. Molecular Systems Biology, 13(11), 952.

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Bacterial Culture and Plasmid Propagation

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Cultures were grown in LB‐Miller broth (BD #2020‐05‐31) for cloning and plasmid propagation. For all other experiments, cultures were grown on a defined industrial minimal medium (MM) containing the following: 5 g/l (NH₄)₂SO₄ (Millipore #AX1385‐1), 5 g/l K₂HPO₄ (VWR #0705), 30 g/l 2‐(N‐morpholino)ethanesulfonic acid (MES; Sigma #M2933), a carbon source as indicated, and a proprietary mixture of micronutrients as previously described (Moser et al, 2012). Carbon sources included glycerol (VWR #97062‐832) or glucose (BDH #8005‐500g). Concentrations of glycerol and glucose here are given as % mass (g/g). Following preparation, the pH of the minimal media was adjusted to 6.8 with NaOH and HCl and the media was filtered through a 0.2 μm filter (Corning #430049). The inducers anhydrotetracycline (aTc; Sigma #37919), 2,4‐diacetylphloroglucinol (DAPG; Santa Cruz Biotechnology #12161‐86‐6), sodium acetate (Sigma #127‐09‐3), and isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; Sigma #367‐93‐1) were added to the concentrations indicated. Antibiotics were added at the following concentrations to maintain plasmids in all liquid cultures and plates unless otherwise indicated: 50 μg/ml kanamycin (GoldBio #25389‐94‐0), 50 μg/ml spectinomycin (GoldBio #22189‐32‐8), 50 μg/ml ampicillin (GoldBio #69‐52‐3), and 35 μg/ml chloramphenicol (GoldBio #25‐75‐7).

Moser F., Espah Borujeni A., Ghodasara A.N., Cameron E., Park Y, & Voigt C.A. (2018). Dynamic control of endogenous metabolism with combinatorial logic circuits. Molecular Systems Biology, 14(11), e8605.

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