Non-native AHLs were synthesized as described previously (Mattmann et al., 2011 (link)). N-(3-oxododecanoyl) L-homoserine lactone (OdDHL) was purchased from Sigma–Aldrich. Chlorophenol red-β-D-galactopyranoside (CPRG) was purchased from Roche.
N 3 oxo dodecanoyl l homoserine lactone
Manufactured by Merck Group
Sourced in United States
N-3-oxo-dodecanoyl-L-homoserine lactone is a chemical compound used in laboratory research. It serves as a signaling molecule involved in quorum sensing, a process of cell-to-cell communication in certain bacteria. The compound plays a role in regulating gene expression and coordinating collective behaviors within bacterial populations.
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Lab products found in correlation
N butyryl dl homoserine lactone, by Merck Group (4 mentions)
N hexanoyl l homoserine lactone, by Merck Group (3 mentions)
N 3 oxo hexanoyl l homoserine lactone, by Merck Group (3 mentions)
N octanoyl l homoserine lactone, by Merck Group (3 mentions)
N 3 oxo octanoyl l homoserine lactone, by Merck Group (3 mentions)
N 3 oxo decanoyl l homoserine lactone, by Merck Group (3 mentions)
N decanoyl dl homoserine lactone, by Merck Group (2 mentions)
N dodecanoyl dl homoserine lactone, by Merck Group (2 mentions)
N 3 hydroxy dodecanoyl dl homoserine lactone, by Merck Group (2 mentions)
N butyryl dl homoserine lactone c4 hsl, by Merck Group (2 mentions)
3 oxo c12 hsl, by Merck Group (2 mentions)
N 3 hydroxytetradecanoyl dl homoserinelactone, by Merck Group (2 mentions)
Chlorophenol red β d galactopyranoside, by Roche (1 mentions)
Formic acid for hplc, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Spectinomycin, by GoldBio (1 mentions)
Thiamine hydrochloride, by Merck Group (1 mentions)
Casamino acid, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by GoldBio (1 mentions)
Neb 10 beta, by New England Biolabs (1 mentions)
12 protocols using n 3 oxo dodecanoyl l homoserine lactone
1
Synthetic AHL Compound Characterization
2
Synthesis and Characterization of AHL Analogs
3
AHL Compound Standards for Quorum Sensing
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The following AHL standards, purchased from University of Nottingham, UK were: N-3-oxo-butyryl-L-homoserine lactone (3OC4-HSL), N-3-hydroxy-butyryl-L-homoserine lactone (3OHC4-HSL), N-3-hydroxy-hexanoyl-L-homoserine lactone (3OHC6-HSL), N-3-hydroxy-octanoyl-L-homoserine lactone (3OHC8-HSL), N-3-hydroxy-decanoyl-L-homoserine lactone (3OHC10-HSL). In addition, N-butyryl-DL-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), N-3-oxo-hexanoyl-L-homoserine lactone (3OC6-HSL), N-octanoyl-L-homoserine lactone (C8-HSL), N-3-oxo-octanoyl-L-homoserine lactone (3OC8-HSL), N-decanoyl-DL-homoserine lactone (C10-HSL), N-3-oxo-decanoyl-L-homoserine lactone (3OC10-HSL), N-dodecanoyl-DL-homoserine lactone (C12-HSL), N-3-oxo-dodecanoyl-L-homoserine lactone (3OC12-HSL), N-3-hydroxy-dodecanoyl-DL-homoserine lactone (3OHC12-HSL), acetonitrile, and formic acid for HPLC were obtained from Sigma-Aldrich (St. Louis, MO, USA).
4
Quorum Sensing Compounds Procurement
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A panel of quorum sensing compounds were purchased from Sigma-Aldrich, Acros or synthesized as previously reported: N-(3-oxododecanoyl)-L-homoserine lactone (C12) (Kravchenko et al., 2008 (link)), N-butyryl-homoserine lactone (C4), N-octanoyl-L-homoserine lactone (C8), 4,5-dihydroxy-2,3-pentanedione (DPD) (Mandabi et al., 2015 (link)), and farnesol. Control compounds were the unnatural R enantiomer of C12, N-(3-oxododecanoyl)-D-homoserine lactone (R-C12), and the lactam analog of C12 (S)-3-oxo-N-(2-oxopyrrolidin-3-yl)dodecanamide (C12-lactam) (Kravchenko et al., 2006 (link)). For receptor labeling experiments, an alkyne-appended diazirine analog of C12 (P6) was synthesized as previously reported by us (Dubinsky et al., 2009 (link)).
5
Quorum Sensing Inhibition by DIM
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QS antagonistic activity of DIM was assessed by comparing the bioluminescence signal observed in wells containing the activated E. coli K802NR or P. aeruginosa pKD-rhlA bioreporter strains carrying the luxCDABE operon with that in the wells containing the same reaction mixture but with the addition of 50 µM DIM. Reaction mixtures comprised either E. coli K802NR and 1.7 nM N-(3-oxododecanoyl)-l -homoserine lactone (3-oxo-C12-HSL) (O9139, SigmaAldrich) or PAO-JP2 (pKD-rhlA) and 1 × 10−5 M N-butyryl-dl -homoserine lactone (C4-HSL) (09945, Sigma-Aldrich). Inhibition was indicated when the addition of DIM to mixtures containing either 3-oxo-C12-HSL or C4-HSL resulted in a reduction in bioluminescence.
6
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.
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%
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-
(Sigma-Aldrich),
(Sigma-Aldrich), gamma-aminobutyric acid (Sigma-Aldrich), N-butyryl-
3-oxohexanoyl-
and N-(3-hydroxytetradecanoyl)-
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.
7
Sodium Lactate and Tilivalline Synthesis
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A filter-sterilized stock solution of 5 mg/mL sodium d -lactate (Sigma-aldrich, Diegem, Belgium) was prepared in serum-free, antibiotics-free DMEM, which was further diluted in serum-free, antibiotics-free DMEM during the experiments to obtain the desired concentrations (100, 500, 1,000, 5,000 µg/mL).
The quorum sensing molecule N-(3-oxododecanoyl)-l -homoserine lactone (Sigma-aldrich, Diegem, Belgium) was dissolved in DMSO to obtain stock solutions of 15, 30, 45 and 60 mM. For experiments, further dilutions (1:500) in DMEM without serum and antibiotics were prepared.
For the synthesis of the pyrrolobenzodiazepine tilivalline, we followed the protocol described by Schneditz et al. (2014 (link)). However, we used the freeze–pump–thaw method (3×) instead of the vacuum/N2 cycle (3×) for degassing the reaction. In this way a yield of 57% was obtained compared to the previously reported 38% (Schneditz et al. 2014 (link)).
For the in vitro experiments, stock solutions of 100, 50, 10, 5 and 1 mM tilivalline were prepared in DMSO, which were further diluted (1:1,000) in DMEM without serum and antibiotics.
The quorum sensing molecule N-(3-oxododecanoyl)-
For the synthesis of the pyrrolobenzodiazepine tilivalline, we followed the protocol described by Schneditz et al. (2014 (link)). However, we used the freeze–pump–thaw method (3×) instead of the vacuum/N2 cycle (3×) for degassing the reaction. In this way a yield of 57% was obtained compared to the previously reported 38% (Schneditz et al. 2014 (link)).
For the in vitro experiments, stock solutions of 100, 50, 10, 5 and 1 mM tilivalline were prepared in DMSO, which were further diluted (1:1,000) in DMEM without serum and antibiotics.
8
Marine Bacteria Quorum Sensing Inhibition
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The marine bacterial strains analysed in this study are summarized in Table 2 . Marine agar or broth (MA or MB respectively) was routinely used to grow marine bacterial strains (Difco, Oxford, UK). SYP agar with 1.5% of sea salt and MA or MB were used in different experiments performed in this work to analyse the potential QSI capability of marine bacteria. All marine bacterial strains used in this study were grown routinely at 23°C. Different N‐Acyl homoserine lactones (AHLs) used in this work were purchased from Sigma‐Aldrich (Cambridge, UK) and were dissolved in DMSO (N‐butyryl‐dl ‐homoserine lactone: C4‐HSL; N‐hexanoyl‐l ‐homoserine lactone: C6‐HSL; N‐(3‐oxooctanoyl)‐l ‐homoserine lactone: 3OC8‐HSL; N‐(3‐oxodecanoyl)‐l ‐homoserine lactone: 3OC10‐HSL; and N‐(3‐oxododecanoyl)‐l ‐homoserine lactone: 3OC12‐HSL).
9
Comparative Analysis of Bacterial Media
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Brain heart infusion (BHI) medium/broth was purchased from Lab M (Heywood, UK), while Luria broth/agar (LB/LA) and Mueller–Hinton broth (MHB) were provided by HiMedia (Mumbai, India). TetraMinTM flakes were provided by Tetra Melle (Melle, Germany). Materials purchased from Sigma-Aldrich (St. Louis, MO, USA) were: resazurin, HHL, 2-heptyl-4-quinolone (HHQ), N-3-oxo-dodecanoyl-L-homoserine lactone (3-oxo-C12-HSL), N-butyryl-L-homoserine lactone (C4-HSL), QA, GA, ChA, Cat, Quer-3-O-Glc, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin mixture, phosphate-buffered saline (PBS), trypsin from porcine pancreas, trypan blue, DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), reference standards of phenolic compounds and tricaine solution. HPLC gradient grade methanol was purchased from J. T. Baker (Deventer, The Netherlands) and p.a. formic acid from Merck (Darmstadt, Germany). All other chemicals (molecular biology grade) were purchased from local companies.
10
AHL-Induced Gene Expression in E. coli
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Experiments were carried out with E. coli TOP10 cells transformed with plasmid constructs (Supplementary Table 3 ). Plasmid constructs were assembled to comply with the BioBrick RCF [10] standard using a variety of molecular cloning techniques. Biological parts were acquired from the registry of Standard Biological parts or from chemical synthesis. DNA sequences of assembled AHL-receiver devices are available for download (10.5281/zenodo.1252305). AHL inducers utilised were purchased as follows: N-Butyryl-DL-homoserine lactone {C4 HSL} (09945 Sigma), 3-oxohexanoyl-l -homoserine lactone {3OC6 HSL} (K3007 Aldrich), N-(3-Oxooctanoyl)-l -homoserine lactone {3OC8 HSL} (O1764 Sigma), N-(3-Oxododecanoyl)-l -homoserine lactone {3OC12 HSL} (O9139 Sigma), N-(3-Hydroxytetradecanoyl)-DL-homoserine lactone {3OHC14 HSL} (51481 Sigma) and N-(p-Coumaroyl)-l -homoserine lactone {pC HSL} (07077 Sigma). AHLs stock solutions were made in 100% DMSO.
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