AHL standards (C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were purchased from the Cayman Chemical Company (Ann Arbor, Michigan, USA). Chromatographic-grade methanol and 99.9% ethyl acetate were purchased from Sigma-Aldrich (Buchs, Switzerland). The standards were dissolved in methanol at a concentration of 1 mM and stored at −20 °C.
C6 hsl
Manufactured by Cayman Chemical
Sourced in United States
C6-HSL is a signaling molecule that is involved in quorum sensing, a process used by bacteria to coordinate gene expression and behavior in response to cell population density. It functions as a chemical communication signal between bacteria.
Automatically generated - may contain errors
Lab products found in correlation
C8 hsl, by Cayman Chemical (3 mentions)
3 oxo c6 hsl, by Cayman Chemical (2 mentions)
3 oxo c10 hsl, by Cayman Chemical (2 mentions)
3 oxo c10 hsl, by Merck Group (2 mentions)
C10 hsl, by Merck Group (2 mentions)
C4 hsl, by Cayman Chemical (2 mentions)
3 oxo c12 hsl, by Merck Group (2 mentions)
Chromatographic grade methanol, by Merck Group (1 mentions)
3 oxo c8 hsl, by Merck Group (1 mentions)
C12 hsl, by Merck Group (1 mentions)
C14 hsl, by Merck Group (1 mentions)
3 oxo c14 hsl, by Merck Group (1 mentions)
N decanoyl homoserine lactone, by Merck Group (1 mentions)
N 3 oxo dodecanoyl l homoserine lactone, by Merck Group (1 mentions)
N 3 oxo hexanoyl l homoserine lactone, by Merck Group (1 mentions)
3 oxo c6 hsl, by Merck Group (1 mentions)
6 protocols using c6 hsl
1
Quantification of Quorum Sensing Signals
2
Cultivation and Characterization of AHL Biosensors
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XY-85 was cultured in marine broth 2,216 (MB; BD Difo™) at 28°C. E. coli strains DH5α and BL21 (DE3) were cultured on Luria–Bertani (LB) agar at 37°C and used as hosts for expressing the protein whose encoding gene was cloned into pET-28a (Ke Lei Biotechnology Co., Ltd). The AHL biosensors Chromobacterium violaceum CV026 and VIR24 (McClean et al., 1997 (link); Someya et al., 2009 (link)), used to detect short-chain (C4 to C8) and long-chain (C8 to C14) AHLs, were maintained on LB agar at 28°C. Pectobacterium caratovorum subsp. caratovorum (Pcc, provided by Dr. Junna He at China Agricultural University) was cultured on Luria–Bertani (LB) agar at 28°C. Kanamycin was added at 50 μg mL−1. C4-HSL, C6-HSL, 3-oxo-C6-HSL, and C8-HSL were purchased from Cayman Chemical Company (Ann Arbor, MI, United States); 3-oxo-C8-HSL, C10-HSL, 3-oxo-C10-HSL, C12-HSL, 3-oxo-C12-HSL, C14-HSL, and 3-oxo-C14-HSL were purchased from Sigma-Aldrich (St. Louis, MO, United States). All of the AHL stock solutions (10 to 500 mM) were prepared in dimethyl sulfoxide (DMSO) and stored under −20°C.
3
Production and Characterization of MomL Protein
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Pectobacterium carotovorum subsp. carotovorum (Pcc) was purchased from the CGMCC (China General Microbiological Culture Collection, Beijing, China) [41 ]. E. coli strain AHL882-5 was used to express the MomL protein. E. coli strain BL21(DE3) was used as a host for protein expression. Proteins were expressed following the cloning of random mutants of the momL gene into pET-24a(+). The strain Chromobacterium violaceum CV026 was used as an indicator in the AHL activity bioassay [42 (link)]. C6-HSL was purchased from the Cayman Chemical Company and prepared in dimethyl sulfoxide (DMSO). M. olearia Th120, CV026 and Pcc were routinely cultured on Luria-Bertani (LB) agar at 28 °C. E. coli strain AHL882-5 was cultured in LB medium at 37 °C. When required, 25 μg/mL kanamycin was added to the solid or liquid media.
4
Bacterial and Fungal Strain Culturing
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Bacterial strains were inoculated with 50 μL (from cryotube) in glass tubes containing 5 mL of MB (Marine Broth, BD Difco) for 24 h at 18°C. For the AHL inhibition tests (short chain-homoserine lactones and long chain-homoserine lactones also called short chain-HSL and long chain-HSL), the bacterial strains were cultured with 1 μM (C6-HSL or 3-oxo-C10-HSL, Cayman Chemical, Ann Arbor, MI, USA) corresponding to the specific biotest being conducted. Fungal strains were inoculated with 3 pieces of agar (1 cm2) from Petri dish cultures. The fungal strains were grown at 18°C for 7 days in 50 mL of PDB 75 % NSW (Potato Dextrose Broth, BD Difco) and liquid MEA 75 % NSW without agar (see previous).
5
Quantifying hchA-smoR Promoter Activity
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To evaluate the expression levels of hchA-smoR promoter, ß-galactosidase assays were performed for the strains E77 wild type and ΔsmoR mutant harboring either the vectors pBBR1MCS-5-PsmoR:lacZ or pBBR1MCS-5-lacZ—the latter used as a control– during growth in LB medium at 30°C, following the protocol described by Miller (1972 ). All bacterial cultures were started with an initial inoculum corresponding to an optical density at 550 nm (OD550) of 0.05. To determine the activity of the hchA-smoR promoter during growth curve, 0.1 ml-samples were taken at different times from 4 to 48 h. To investigate the effect of the presence of AHL molecules in the activity of the hchA-smoR promoter, initial cultures were supplemented with various synthetic AHLs (Cayman Chemical) –C6-HSL, oxo-C8-HSL, C8-HSL, and C10-HSL– with different concentrations (1 up to 10 μM), and 0.1 ml samples were taken and measured after 24 h and 48 h of incubation at 30°C. After analyzing the data we determined β-galactosidase specific activities in Miller Units (Miller, 1972 ). All AHL stocks were solubilized in 70% acetonitrile/water acidified with 0.1 M HCl final concentration. All experiments were performed by triplicate and comparison of ß-galactosidae activity was performed by One-Way analysis of variance (ANOVA) with a Bonferroni's multiple comparison post-test.
6
Degradation of Quorum Sensing Signals by Bacillus spp.
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The AHLs degradation activity of cell-free lysates of Bacillus spp. isolates 30b, 32C, and 6 was assessed against different AHL signals using the well diffusion assay. AHL signals tested were; 100 µM C4-HSL (Cayman), 50 µM C6-HSL, 50 µM N- decanoyl-homoserine lactone (C10-HSL), 25 µM N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), 25 µM N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C10-HSL), 25 µM 3-oxo-C12-HSL (Sigma-Aldrich). The relative degradation activity was calculated for each AHL signal by calculating the residual AHL from a calibration curve constructed using different concentrations of standard AHL and their induced zone diameter using two AHLs reporter strains; Chromobacterium violaceum (C. violaceum) CV026 and A. tumafaciens KYC55, which respond to short chain and medium-to-long chain AHLs, respectively.39 (link)–42 The maximum degrading activity is defined as 100% relative degradation activity.43 (link)
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