The bacterial strains and plasmids used in this study are listed in Table 1 . All bacteria were cultured in lysogeny broth (LB; 10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) or on LB agar (15 g/l agar) at 37°C except Serratia plymuthica, which was grown at 30°C. Media were supplemented with the following chemicals (Applichem, Darmstadt, Germany) when appropriate: 5 g/l glucose; 100 μg/ml ampicillin (Ap); 200 μg/ml carbenicillin (Cb); 30 μg/ml chloramphenicol (Cm); 5 μg/ml gentamicin (Gm); 10 μg/ml tetracycline (Tc); 50 μg/ml kanamycin (Km); and 1 mM isopropyl-β-D -thiogalactopyranoside (IPTG). Plasmids pTrc99A and pTrc99A-Ptrc-budAB were introduced into the mixed-acid fermenters by electroporation. All oligonucleotides used in this work are listed in Table 2 , and were purchased from IDT (Haasrode, Belgium).
Tetracycline
Manufactured by AppliChem
Sourced in Germany
Tetracycline is a type of laboratory equipment used in scientific research and analysis. It is primarily used for the detection and quantification of the tetracycline class of antibiotics in various samples, such as pharmaceutical products, food, or environmental samples. The core function of this equipment is to provide accurate and reliable measurements of tetracycline concentrations, which is crucial for quality control, regulatory compliance, and research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Ampicillin, by AppliChem (4 mentions)
Gentamicin, by AppliChem (3 mentions)
Kanamycin, by AppliChem (3 mentions)
Chloramphenicol, by AppliChem (2 mentions)
Polymyxin b, by Merck Group (2 mentions)
Melittin, by Enzo Life Sciences (2 mentions)
Carbenicillin, by AppliChem (1 mentions)
Luria bertani medium, by BD (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Kanamycin, by NZYTech (1 mentions)
X gal, by NZYTech (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by NZYTech (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Carl Roth (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by AppliChem (1 mentions)
Ahringer library, by Source Bioscience (1 mentions)
10 protocols using tetracycline
1
Bacterial Strains and Plasmid Protocols
2
Construction of MRSA-Derived Isogenic MSSA Strain
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Bacterial strains and plasmids used in this study are listed in Table 1 . Escherichia coli strain DH5α was routinely used for plasmid propagation and cloning experiments, and cultivated on Luria-Bertani (LB) medium (Becton–Dickinson, Sparks, MD, USA) supplemented with 100 mg/L ampicillin (AppliChem, Darmstadt, Germany) at 37 °C. S. aureus strains were grown with aeration in trypticase soy broth (TSB) (Difco Laboratories, Detroit, MI, USA) in a rotating incubator (at 180 rpm) at 37 °C. If required, tetracycline and kanamycin (AppliChem) were added at a final concentration of 10 mg/L. Oxacillin (Sigma-Aldrich, Buchs, Switzerland) was used at 4 mg/L, which corresponded to the MICs for strain N315.
Bacterial strains and plasmids
| Strain or plasmid | Relevant characteristics | References |
|---|---|---|
| Strains | ||
| E. coli DH5α | Host for DNA cloning | Laboratory collection |
| S. aureus | ||
| RN4220 | Restriction-deficient derivative of S. aureus RN450 | [11 (link)] |
| N315 | MRSA carrying type II SCCmec | [12 (link)] |
| N315EX | Isogenic MSSA derivative of N315 | This study |
| Plasmids | ||
| pUC28 | ColE1 replicon, high copy number vector for cloning, ampicillin resistance | [13 (link)] |
| pSR3-1 | Thermosensitive-replicon plasmid carrying the ccrAB genes of strain N315 (used for SCCmec excision in N315), tetracycline resistance | [7 (link)] |
| qPlasmid | Plasmid used for qPCR analysis | This study |
3
Bacmid Recombination and Baculovirus Production
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For bacmid recombination [51 ], chemically competent DH10EmBacY cells were transformed with the expression construct of interest and incubated overnight in the shaker at 37°C in 2 mL of Luria-Bertani (LB) medium. Culture was plated on LB agar plates containing 50 μg/mL kanamycin (NZYTech), 10 μg/mL tetracycline (AppliChem), 10 μg/mL gentamycin (Sigma-Aldrich), 0.5 mM IPTG (NZYTech) and 40 μg/mL X-Gal (NZYTech), and incubated at 37°C. White colonies containing recombined bacmid were picked and cultured overnight in LB medium supplemented with kanamycin, tetracycline, and gentamycin in the shaker at 37°C. Bacmid DNA was isolated by alkaline lysis followed by isopropanol precipitation. For virus production [51 ], Sf21 cells in 6-well plates (106 cells/well) were transfected with bacmid DNA using X-tremeGene HP DNA Transfection Reagent (Roche). 50 hr after transfection, the supernatant containing initial virus V0 was collected and used to infect a 25-mL culture (SFM4 medium, Hyclone) of Sf21 cells (0.5 × 106 cells/mL) to produce V1 virus. Culture density was kept at 1 × 106 cells/mL (diluted with fresh medium when needed) until cell proliferation ceased. 48 hr after proliferation arrest, V1 was harvested by centrifuging the cell suspension (5 min at 800 × g) and collecting the supernatant. V1 stock was stored at 4°C in the dark.
4
C. elegans Lifespan Assay with RNAi
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Nematodes were grown and maintained at 20°C; ambient temperatures during handling (transfer) of worms were between 20 and 25°C. Maintenance was on nematode growth medium (NGM) agar plates spotted with E. coli OP50 as food source, as described elsewhere [46 (link)]. Synchronization was performed by washing, followed by centrifugation to separate the eggs from the nematodes. Eggs were transferred to fresh NGM agar plates (Carl Roth, 6494) and allowed to hatch and grow for 64 h. Lifespan assays with C. elegans-specific RNA interference were conducted as previously described [47 (link)]. Briefly, young adult worms, 64 h after synchronization, were transferred to NGM agar plates containing 1 mM IPTG (Thermo Fisher, R0392), 100 µg/ml ampicillin (Carl Roth, K029) and, if necessary, 12.5 μg/ml tetracycline (AppliChem, A2228). Plates were spotted with E. coli HT115 (DE3) containing L4440 empty vector (Addgene, 1654) or L4440 containing a f53e2.1, y39h10a.6, t07d1.2 or y54e5a.7 DNA fragment. For the first 10 d, nematodes were transferred to fresh plates daily; thereafter, they were transferred every other day. Experiments were performed in quintuplicates and two independent times. Worms showing no movement, no reaction to gentle stimulation and no pharyngeal pumping were scored as dead. Worms lost or disintegrated due to internal hatchings were censored.
5
RNAi Gene Knockdown in C. elegans
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For RNAi gene knock-down experiments, we applied E. coli HT115 to the worms. Clones for RNAi against aat-1 and F21D5.1 were derived from Ahringer library (Source BioScience, Nottingham, UK) and C. elegans ORF Collection (Thermo Scientific, Waltham, MA, USA) (Supplementary Table 1 ) respectively, and were sequenced prior use. The bacteria were spotted on NGM plates containing additionally 1 mM IPTG, 100 μg ml−1 Ampicillin and, if required, 12.5 μg ml−1 tetracycline (all from Applichem, Darmstadt, Germany).
6
Bacterial Growth Curves and Antibiotic Susceptibility
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Bacterial growth curves were determined by inoculating a single colony each of AWS-003 (Δtdk; referred to as the wild-type in Fig. 4 ) and AWS-029 (∆masB) into 5 ml TYG medium and incubating overnight under anaerobic conditions. These cultures were sub-cultured (1:100 dilution) in 2 ml TYG medium containing the indicated final concentrations of doxycycline (Sigma-Aldrich), tetracycline (AppliChem) or a water control. The samples (200 µl volume) were incubated in a 96-well flat-bottom plate (Nunclon) at 37 °C (doxycycline) or 40 °C (tetracycline) with continuous shaking (double orbital) in a microplate spectrophotometer (BioTek Epoch 2). Optical densities were recorded every 20 min. The assay was performed in three biological replicates, each comprising technical duplicates.
Antibiotics strip assays were performed by dipping a sterile cotton swab into overnight TYG cultures of AWS-003 or AWS-029 and streaking on BHIS agar plates containing strips for doxycycline (EM103 (HiMedia; in Fig.4c ) or 92156 (Liofilchem; in Fig. 5g )) or tetracycline (EM056; HiMedia). The plates were incubated anaerobically for 48 h at 37 °C and images were taken. The minimal inhibitory concentrations were derived from the positions where the inhibition ellipses intersected the strips.
Antibiotics strip assays were performed by dipping a sterile cotton swab into overnight TYG cultures of AWS-003 or AWS-029 and streaking on BHIS agar plates containing strips for doxycycline (EM103 (HiMedia; in Fig.
7
Antibiotic Susceptibility Profiling of LAB
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The microdilution-broth assay was performed in order to assess the susceptibility pattern of isolates to common antibiotics. The selected isolates were tested for their resistance to various antibiotics according to the technical guidelines of the ISO [17] . Ampicillin, gentamycin, kanamycin, streptomycin, tetracycline, chloramphenicol, and erythromycin (purchased from Applichem, Spain) were used at concentrations ranging from 0.5 to 1024 mg/L. Each well was filled with 100 mL of diluted inoculum and 100 mL of antibiotic solution using LAB susceptibility test medium (LSM, HiMedia Laboratories, Mumbai, India), and plates were incubated at 37 °C for 48 h. Microorganisms' growth was visually monitored, and the minimum inhibitory concentration (MIC) value of each antibiotic was determined as the lowest concentration able to inhibit visible cell growth. The MIC cut-off values established by the European Food Safety Authority [18] were taken into consideration in order to classify LAB isolates as susceptible or resistant to each antibiotic that was tested.
8
Retro-inverse TAT-RasGAP peptide characterization
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The retro-inverse TAT-RasGAP317-326 peptide (amino acid sequence DTRLNTVWMWGGRRRQRRKKRG) and its N-terminal FITC-labeled derivative were provided by SBS Genetech (Beijing, China) and stored at −20°C. Melittin was provided by Enzo Life Science (Farmingdale, NY), polymyxin B, meropenem and aztreonam by Sigma-Aldrich (Burlington, MA), and gentamicin and tetracycline by Applichem (Darmstadt, Germany).
9
Antimicrobial Retro-Inverso Peptide TAT-RasGAP
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TAT-RasGAP317-326 is a retro-inverso peptide (i.e. reversed direction compared to natural sequence including D-amino acids) with an antimicrobial activity [20] composed of amino acids 48-57 of HIV TAT protein (RRRQRRKKRG) and 317-326 of human RasGAP protein (DTRLNTVWMW) linked with two glycines. TAT-RasGAP317-326 was synthesized by SBS Genetech (Beijing, China). Ciprofloxacin, tetracycline and gentamicin were from Applichem (Darmstadt, Germany), rifampicin and polymyxin B from Sigma-Aldrich (Saint-Louis, MO), and melittin from Enzo Life Sciences (Farmingdale, NY).
10
Proliferation Assay for Flp-In T-REx 293 and PANC-1 Cells
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For proliferation assay 1x 10 3 Flp-In T-REx 293 cells or 2.5 3 10 3 PANC-1 cells were seeded without any antibiotics in 96-well plates in DMEM without pyruvate and monitored five days under standard conditions. 96-well plates used for Flp-In T-REx 293 cells were coated with 50 mg/ml Poly-D lysine (Sigma Aldrich) in PBS before. METTL8-F/H expression in Flp-In T-REx À293 cells was induced by 1 mg/ml tetracycline (AppliChem). After desired incubation time, cells were fixed with 50 ml crystal violet solution (0.5% [w/v] crystal violet in 20% [v/v] MeOH) at RT for 10 minutes. Fixed cells were washed twice with 100 ml warm water followed by two further PBS washing steps of two minutes. Lastly cells were rinsed with water and air-dried. Cells were quantified by crystal violet intensity detected at 590 nm using a Multimode-Microplate reader Mithras LB 940 (Berthold Technologies). Crystal violet was eluted from the cells before by 50 ml 0.1 M sodium citrate in 50% [v/v] EtOH.
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