Minimum inhibitory concentrations (MICs) of AHL-degrading bacteria were determined using microtiter plate dilution assays in R2A broth with about 1 × 105 cells/well, as previously described (Yajko et al., 1987 (link); Riesenfeld et al., 2004 (link)). MICs were determined after 1, 2, and 3 days of incubation at 30°C in the dark. MICs were read using a Multiskan® Spectrum microplate spectrophotometer (Thermo Labsystems, Vantaa, Finland) and was defined as the lowest concentration of an antimicrobial agent at which the organism showed no visible growth (Yajko et al., 1987 (link)). E. coli strain EPI300TM (Epicentre, Madison, WI, United States) was used as a negative control. The criteria used for the interpretation of antimicrobial susceptibility were based upon the achievable levels of antimicrobial agents. The tested antibiotics were PENG, AMP, AMO, carbenicillin (CAR), piperacillin (PIP), CEFL, and CEFD. All these antibiotics are in common use, and the environmental samples used in the present study were polluted with wastewater from PENG, AMP, AMO, CEFL, and CEFD production. The antibiotics were tested at concentrations of 8, 16, 32, 64, 125, 250, and 500 μg/mL.
Epi300
Manufactured by Illumina
Sourced in United States
The EPI300 is a laboratory equipment product designed for the isolation and amplification of small DNA fragments from complex samples. It functions as an efficient method for extracting and amplifying target DNA sequences from various sources, enabling researchers to obtain high-quality genetic material for further analysis and study.
Automatically generated - may contain errors
Lab products found in correlation
Multiskan spectrum microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Zero blunt topo pcr cloning kit for sequencing, by Thermo Fisher Scientific (1 mentions)
Pcc1fos, by Illumina (1 mentions)
Bl21 de3, by Takara Bio (1 mentions)
One glo, by Promega (1 mentions)
Nabut, by Merck Group (1 mentions)
Quanti blue, by InvivoGen (1 mentions)
Infinite 200, by Tecan (1 mentions)
Pcc2fos vector, by Illumina (1 mentions)
Lycopene standard, by Merck Group (1 mentions)
High fidelity dna polymerase kod plus neo, by Toyobo (1 mentions)
Phis2, by Takara Bio (1 mentions)
16 protocols using epi300
1
Determining MICs of AHL-Degrading Bacteria
2
Stylissa massa Metagenome Library Construction
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The marine sponge, Stylissa massa, was collected from the offshore of Ishigaki island, Okinawa, Japan. The E. coli strains, EPI300 TM (Epicentre Biotechnologies) were used in metagenome library construction, DH5α (TOYOBO) and EC100 TM (Epicentre Biotechnologies) in cloning and BL21 (DE3) (Novagen) in recombinant protein expression, respectively. The plasmid pCC1FOS (Epicentre Biotechnologies) was used for metagenome library construction and in standard cloning procedures. The Zero Blunt TOPO PCR Cloning Kit for Sequencing (Life Technologies) was used for the cloning of PCR amplicons, and the pET25b (Novagen) vector was used for protein expression.
3
E. coli Culture Conditions and Strains
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E. coli cultures were grown at 37°C on Luria-Bertani (LB) agar or in LB broth supplemented with the appropriate antibiotics. The following antibiotic concentrations were used for the E. coli strains: chloramphenicol, 12.5 μg mL−1; ampicillin, 100 μg mL−1. E. coli strain EPI300 was obtained from Epicentre (Madison, WI, USA). E. coli strain BL21 (DE3) was obtained from Takara Bio (Otsu, Japan).
4
Automated High-Throughput Cellular Assays
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All experiments were performed using a robotic pipetting workstation (Hamilton Robotics Star Line) for high seeding, stimulation and revelation homogeneity. For each experiment, cells were seeded at 25000 cells/well in 96 well-plates and incubated 24 h prior to activation. Homogeneous cellular activations were performed using sodium butyrate (NaBut, 2 mM, Sigma) for PPARγ and ANGPTL4 reporter systems or 0,45 µm-filtered, sonicated E. coli (Epi300 from Epicentre, 10% vol/vol) for NFκB-SEAP system. Cells were then incubated 24 h prior quantification of the reporter gene expression (alkaline phosphatase (SEAP) or luciferase). SEAP (for NFκB system) secretion or intracellular luciferase expression (for both ANGPTL4 and PPARγ systems) were revealed using the QUANTI-Blue (Invivogen) or One-Glo (Promega) reagent respectively, according to the manufacturers’ instructions. All measurements were performed using a microplate reader (Infinite 200, Tecan) and quantified at 655 nm OD for SEAP and by luminescence for luciferase.
5
High-Andean Soil Metagenomic Library Screening
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The metagenomic library, which consists of 18,432 clones, was constructed using DNA extracted from high-Andean forests soils from the National Natural Park “Los Nevados [17] . DNA was purified using the Ultra Clean Mega Soil DNA kit (MoBio) and fragments of approximately 30 kb were ligated to the pCC2FOS vector (Epicentre) and used to transform Escherichia coli EPI300™, following the manufacturer’s indications (Epicentre). The identification of positive clones for cellulose degradation was done as published [17] . Briefly, clones capable of growing on minimum salt medium (MM; 0.2% NaNO3, 0.1% K2HPO4, 0.05% MgSO4, 0.05% KCL, 0.02% Peptone, 12.5 μg/ml chloramphenicol) with pretreated OPEFB as only source of carbon for 35 days at 30 °C and 200 rpm, were presumed to be cellulases carriers. Then, these colonies were enriched in Luria-Bertani (LB) medium for 2 days and transferred to a solid MM containing carboxymethyl cellulose. To detect cellulose hydrolysis, Congo red staining was used and the presence of a hydrolysis halo surrounding a colony was taken as a positive clone for cellulose degradation.
6
Genetic Engineering of Streptomyces sp. 4F
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E. coli strain DH5α was used for cloning of plasmids with pUC replication origin (pUCori); strain EPI300 (Epicentre) was used for cloning oriV-containing plasmids; and strain ET12567 was used for conjugation with Streptomyces sp. 4F [26] (link). E. coli strains were grown in Luria-Bertani medium [27] (link), while Streptomyces sp. 4F was grown on mannitol soya flour [28] (link) plates for spore preparation and on R2YE plates [29] for actinorhodin production.
Enzymes used in this study (unless specified) were purchased from NEB. GeneRuler 1-kb DNA ladder was purchased from Thermo Fermentas and λ-HindIII ladder was from TAKARA. High fidelity DNA polymerase KOD Plus Neo was purchased from TOYOBO. The lycopene standard was purchased from Sigma-Aldrich.
Primers used in this study were listed inTable S1
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E. coli strain DH5α was used for cloning of plasmids with pUC replication origin (pUCori); strain EPI300 (Epicentre) was used for cloning oriV-containing plasmids; and strain ET12567 was used for conjugation with Streptomyces sp. 4F [26] (link). E. coli strains were grown in Luria-Bertani medium [27] (link), while Streptomyces sp. 4F was grown on mannitol soya flour [28] (link) plates for spore preparation and on R2YE plates [29] for actinorhodin production.
Enzymes used in this study (unless specified) were purchased from NEB. GeneRuler 1-kb DNA ladder was purchased from Thermo Fermentas and λ-HindIII ladder was from TAKARA. High fidelity DNA polymerase KOD Plus Neo was purchased from TOYOBO. The lycopene standard was purchased from Sigma-Aldrich.
Primers used in this study were listed in
7
Construction of Metagenomic Libraries
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8
Cloning and Screening of CRISPR sgRNAs
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E. coli Epi300 (F’λ−mcrAΔ(mrr-hsdRMS-mcrBC)ϕ80dlacZ Δ M15 Δ (lac)X74 recA1 endA1 araD139 Δ (ara,leu)7697 galU galK rpsL (StrR) nupG trfA dhfr) (Epicenter) was used for cloning the sgRNA pools. Screening sgRNA activity using a two-plasmid enrichment was done in NEB 5-alpha F’IqE. coli (F’ proA+B+lacIqΔ(lacZ) M15 zzf::Tn10(TetR) /fhuA2 Δ(argF-lacZ)U169 phoA glnV44ϕ80Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17) strain harboring pTox. Citrobacter rodentium DBS100 was used for screening of sgRNA activity against chromosomal targets.
9
Construction of Bacterial Cloning Vectors
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PCR primers to generate the first intermediate vector contained I-SceI sites and ClaI sites. The PCR product was ClaI digested, self-ligated and propagated in E. coli DH5α.
To generate the second intermediate vector, two NsiI sites were first introduced into pcc2FOS by overlapping PCR and subcloning. Next, a kanR fragment was amplified from pHis-2 (Clontech) using primers containing a HindIII site, and then subcloned into the NsiI-pcc2FOS at the HindIII site. The LacZ-kanR-LacZ fragment of the intermediate vector was PCR amplified using primers containing 20-nt of random sequence and an NheI site or ClaI site. The PCR product was NheI and ClaI digested, subcloned into the first intermediate vector, and then transformed into E. coli strain EPI300 (Epicentre) using electroporation (Bio-Rad). Transformants were cultured on LB plates with 50 μg/ml kanamycin and 12.5 ug/ml chloramphenicol over night before counting and collecting.
To generate the second intermediate vector, two NsiI sites were first introduced into pcc2FOS by overlapping PCR and subcloning. Next, a kanR fragment was amplified from pHis-2 (Clontech) using primers containing a HindIII site, and then subcloned into the NsiI-pcc2FOS at the HindIII site. The LacZ-kanR-LacZ fragment of the intermediate vector was PCR amplified using primers containing 20-nt of random sequence and an NheI site or ClaI site. The PCR product was NheI and ClaI digested, subcloned into the first intermediate vector, and then transformed into E. coli strain EPI300 (Epicentre) using electroporation (Bio-Rad). Transformants were cultured on LB plates with 50 μg/ml kanamycin and 12.5 ug/ml chloramphenicol over night before counting and collecting.
10
PCR Cloning and Transformation
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PCR primers contained 20-nt of random sequence flanked by cloning sites at the 5΄ ends and sequence complementary to the pcc2FOS vector at the 3΄ ends. PCR products were NheI digested, self-ligated and transformed into Escherichia coli strain EPI300 (Epicentre) using electroporation (Bio-Rad). Transformants were cultured on LB plates containing 12.5 μg/ml chloramphenicol over night before counting. The clones were washed off plates into liquid LB, pooled together, and stored at −80°C with glycerol.
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