On day 1, yeast scFv libraries, as well as germline and somatic clonal strains, were thawed by inoculating 5 mL SDCAA (1.71 g/L YNB without amino acids and ammonium sulfate (Sigma-Aldrich #Y1251), 5 g/L ammonium sulfate (Sigma-Aldrich #A4418), 2% dextrose (VWR #90000–904), 5 g/L Bacto casamino acids (VWR #223050), 100 g/L ampicillin (VWR # V0339)) with 150 μL glycerol stock (saturated culture with 5% glycerol) and rotated at 30°C for 20 hr. On day 2, yeast cultures were back-diluted to OD600 = 0.2 in 5 mL SDCAA and rotated at 30°C for approximately 4 hr, or until reaching log phase (OD600 = 0.4–0.8). 1.5 mL log-phase cells were then pelleted, resuspended in 4 mL SGDCAA (1.71 g/L YNB without amino acids and ammonium sulfate (Sigma-Aldrich #Y1251), 5 g/L ammonium sulfate (Sigma-Aldrich #A4418), 0.2% dextrose (VWR #90000–904), 1.8% galactose (Sigma-Aldrich #G0625), 5 g/L Bacto casamino acids (VWR #223050), 100 g/L ampicillin (VWR #V0339)), and rotated at room temperature for 20–22 hr.
Ampicillin
Manufactured by Avantor
Sourced in United States, China, Spain
Ampicillin is a broad-spectrum antibiotic commonly used in laboratory settings. It is a type of penicillin that inhibits the synthesis of bacterial cell walls, effectively preventing the growth and proliferation of various bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Kanamycin, by Avantor (15 mentions)
T4 dna ligase, by Takara Bio (7 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (6 mentions)
Vancomycin, by Avantor (5 mentions)
Chloramphenicol, by Avantor (5 mentions)
Chloromycetin, by Avantor (5 mentions)
Plasmid mini prepare kit, by Corning (5 mentions)
Dna mini kit, by Corning (5 mentions)
Bacto casamino acids, by Avantor (4 mentions)
Pmd19 t plasmid, by Takara Bio (4 mentions)
Ex taq dna polymerase, by Takara Bio (4 mentions)
Escherichia coli dh5α, by Takara Bio (4 mentions)
Isopropyl β d thiogalactopyranoside, by Avantor (4 mentions)
Erythromycin, by Avantor (3 mentions)
Ampicillin, by Merck Group (3 mentions)
Tetracycline, by Merck Group (3 mentions)
Imidazole, by Merck Group (3 mentions)
Restriction enzyme, by Thermo Fisher Scientific (3 mentions)
Neomycin sulfate, by Avantor (3 mentions)
Gentamicin, by Avantor (3 mentions)
64 protocols using ampicillin
1
Yeast scFv Library Expression
2
Recombinant Expression of TaBgl2
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Codon optimization, subsequent synthesis, cloning into plasmid pET-20b(+) and transformation of TaBgl2 gene into host Escherichia coli BL21(DE3) were carried out by GenScript (Hong Kong, China) Limited (Hong Kong, China). Restrictions sites BamHI and HindIII on multiple cloning site of plasmid pET-20b(+) were selected for insert cloning with the resultant plasmid annotated as pET-20b(+)-bgl2. Transformants were confirmed by selective growth on LB agar plates supplemented with 100 μg/mL ampicillin (Amresco, Solon, OH, USA), with subsequent sequencing procedures using T7 terminator primer on extracted plasmid template that were carried out by Apical Scientific Sdn. Bhd. (Seri Kembangan, Selangor, Malaysia).
E. coli strains harboring expression vector for TaBgl2 were grown in 50 mL of Terrific Broth liquid medium (24 g/L yeast extract, 12 g/L tryptone, 4 mL/L glycerol, 100 mL/L potassium phosphate solution containing 0.17 M KH2PO4 and 0.72 M K2HPO4) supplemented with 100 μg/mL ampicillin (Amresco, Solon, OH, USA) in 250-mL flasks on a rotary shaker (150 rpm) at 37 °C. Cultivation then continued for 14 h with temperature reduced to 25 °C once absorbance at 660 nm reached 0.7. Resultant cultures were later separated, with the fractions designated as ‘extracellular’, ‘periplasm’, and ‘cytoplasm’, respectively.
E. coli strains harboring expression vector for TaBgl2 were grown in 50 mL of Terrific Broth liquid medium (24 g/L yeast extract, 12 g/L tryptone, 4 mL/L glycerol, 100 mL/L potassium phosphate solution containing 0.17 M KH2PO4 and 0.72 M K2HPO4) supplemented with 100 μg/mL ampicillin (Amresco, Solon, OH, USA) in 250-mL flasks on a rotary shaker (150 rpm) at 37 °C. Cultivation then continued for 14 h with temperature reduced to 25 °C once absorbance at 660 nm reached 0.7. Resultant cultures were later separated, with the fractions designated as ‘extracellular’, ‘periplasm’, and ‘cytoplasm’, respectively.
3
Operant Self-Administration of Alcohol in Rats
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Rats were anesthetized with isoflurane (Henry Schein Medical) at a rate of 4% for induction and 2% for maintenance at an oxygen flow rate of 1 L/min. Surgery was conducted as previously described (Wright et al., 2017 (link)), with minor modifications. Vascular access buttons were connected to indwelling intravenous SILASTIC catheters (Instech Laboratories), which were inserted into the right jugular vein. Catheters were flushed twice daily with 0.1 ml heparinized saline (50 U/ml; Alfa Aesar) and ampicillin (30 mg/ml; VWR). Rats were given a 3 d recovery period before operant self-administration testing. Catheter patency was tested by intravenously administering 0.05 ml of xylazine (2.5 mg/ml) on days where rats were not exposed to alcohol or ketamine (Sunday) each week during the self-administration period. If rapid loss of muscle tone was not observed, the catheter was no longer considered patent, and a new catheter was inserted into the left jugular vein. If a rat failed a second patency test, the rat was removed from the study and not included in statistical analyses. We excluded some rats because of the failure of catheter patency (n = 2) during operant self-administration. The two excluded rats included one high-alcohol intake male that self-administered saline and one high-alcohol intake female that self-administered saline.
4
Modulation of Intestinal Microbiota in Mice
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Il10-/- mice were weaned 21 days after birth and supplemented with a 5 mM solution of either 2-fucosyllactose (2FL), 3-fucosyllactose (3FL), 3-sialyllactose (3SL), 6-sialyllactose (6SL) (Glycom A/S, Hørsholm, Denmark) or D-lactose (Sigma-Aldrich, Buchs, Switzerland) in sterile filtrated water ad libitum for 28 days. For depletion of intestinal microbiota, 4–6 weeks old mice were supplied with 0.5 g/L vancomycin (AppliChem), 1 g/L ampicillin (VWR), 1 g/L neomycin (Fisher Bioreagents) and 0.2% aspartame (Sigma) in drinking water for 6 days. Once per day, mice received intragastric gavage of 250 μL of a 1 g/L metronidazole (Sigma) solution. After antibiotic treatment, mice were treated with 10% PEG 3000 solution (Sigma) in drinking water to flush the remaining bacteria and antibiotic. Neo-colonization with specific bacterial strains was done by intragastric gavage of 250 μL containing 109 CFU of the respective strain in PBS. Fecal pellets were collected to monitor changes of the microbiota.
5
Yeast RBD Library Expansion
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First, we thawed yeast RBD libraries, as well as Wuhan Hu-1 and Omicron BA.1 clonal strains, by inoculating 150 μL of corresponding glycerol stock (saturated culture with 5% glycerol stored at −80 °C) in 5 mL SDCAA at 30 °C for 20 hr. On the next day, yeast cultures were diluted to OD600=0.67 in 5 mL SGDCAA (6.7 g/L YNB without amino acid VWR #90004-150), 5 g/L ammonium sulfate (Sigma-Aldrich #A4418), 2% galactose (Sigma-Aldrich #G0625), 0.1% dextrose (VWR #90000–904), 5 g/L Bacto casamino acids (VWR #223050), 1.065 g/L MES buffer (Cayman Chemical, Ann Arbor, MI, #70310), 100 g/L ampicillin (VWR # V0339)), and rotated at room temperature for 16–20 hr.
6
Antimicrobial Susceptibility of Streptococcus Species
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S. pyogenes, S. agalactiae, and S. salivarius were tested for their antimicrobial susceptibility by broth microdilution. Ampicillin (Cat # 97061–442) was obtained from VWR (Amresco) and Erythromycin (Cat # TCE0751-5G) was obtained from VWR (TCI). Bacteria were maintained on blood agar plates at 37 °C, and single colonies selected for inoculation into liquid overnight cultures in sterile Brain-Heart Infusion (BHI, VWR Cat # 90003–038). For each culture, 14 ml of BHI media was inoculated in a sealed 15 ml falcon tube for overnight incubation at 37 °C (no shaking). Gentle inversion was used to mix the cultures prior to setting up the assay.
For the experiment, 5 μl of overnight culture was mixed with 5 ml of BMI media (1000x dilution) in a sterile tray and gently mixed. This dilute culture was added at 180 μl per well of a 96-well plate pre-loaded with 20 μl of antibiotic solutions ranging, for Erythromycin, from 0.5 to 32 μg/ml (10x) to produce the desired final concentrations of 0.05–3.2 μg/ml. For Ampicillin, the stocks were 1.25 μg/ml-80 μg/ml resulting in final concentrations of 0.125 μg/ml-8 μg/ml. The 96-well plate was then transferred to a FilterMax F5 microplate reader for a 20 h incubation at a temperature of 37 °C, with readings taken every 30 min. A 10-s orbital shaking was performed prior to each reading.
For the experiment, 5 μl of overnight culture was mixed with 5 ml of BMI media (1000x dilution) in a sterile tray and gently mixed. This dilute culture was added at 180 μl per well of a 96-well plate pre-loaded with 20 μl of antibiotic solutions ranging, for Erythromycin, from 0.5 to 32 μg/ml (10x) to produce the desired final concentrations of 0.05–3.2 μg/ml. For Ampicillin, the stocks were 1.25 μg/ml-80 μg/ml resulting in final concentrations of 0.125 μg/ml-8 μg/ml. The 96-well plate was then transferred to a FilterMax F5 microplate reader for a 20 h incubation at a temperature of 37 °C, with readings taken every 30 min. A 10-s orbital shaking was performed prior to each reading.
7
Synthesis and Characterization of Novel Compounds
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Lithocholic acid (95%, Aldrich), lithium aluminum hydride (95%, Aldrich), 2-bromoacetyl chloride (95%, Aldrich) and 6-bromohexanoyl chloride (97%, Aldrich) were used as received. N,N,N′,N′-tetramethyl-1,2-ethanediamine (99%, TCI) and N,N,N′,N′-tetramethyl-1,6-hexanediamine (99%, Aldrich) were distilled before use. Tetrahydrofuran (THF) and N, N-dimethylformamide (DMF) were dried over drying columns. Ampicillin was purchased from VWR as the pure form. All other reagents and solvents were from commercial resources and used as received unless otherwise mentioned. Spectrum Spectra/Por® 3 Dialysis Membranes with MWCO 3500 were purchased from VWR. All other reagents used for biological assays were purchased from Thermo Fisher Scientific or Sigma Aldrich and will be mentioned in the respective sections.
8
Yeast Library Induction and Expansion
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On day 1, the yeast CH65 library and isogenic strains containing the pCHA-UCA860 or pCHA-CH65 plasmids were thawed by inoculating 5 mL SDCAA with 150 µL glycerol stock and rotating at 30°C for 24 hr. On day 2, yeast cultures were back-diluted to OD600 = 0.2 in 5 mL SDCAA and rotated at 30°C until they reached an OD600 = 0.4–0.6 (about 4 hr). Subsequently, 1.5 mL of these log-phase cultures were pelleted, resuspended in 4 mL SGDCAA (1.71 g/L YNB without amino acids and ammonium sulfate [Sigma-Aldrich #Y1251], 5 g/L ammonium sulfate [Sigma-Aldrich, #A4418], 1.8% galactose [Sigma-Aldrich #G0625], 0.2% dextrose [VWR #90000-904], 5 g/L Bacto casamino acids [VWR #223050], 100 g/L ampicillin [VWR # V0339], 5.4 g Na2HPO4 [Sigma-Aldrich, #S7907], 8.56 g NaH2PO4.H2O [Sigma-Aldrich, #S9638]) (Chao et al., 2006 (link)), and rotated at room temperature for 20–22 hr.
9
Scalable RNAi Library Preparation
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OrthoList RNAi plates were replicated in LB agar, and bacteria were grown overnight at 37 °C. The advantage of growing the bacteria in solid media is the ease of visualising the clones where the bacteria did not grow. If needed, the samples can be kept at 4 °C for 2 days maximum. Next day, we inoculated the RNAi library in 2.2-ml 96-well plates (VWR). Using the pin replicator, we inoculated the bacteria from the LB agar into 1.2 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich). Positive and negative controls were added in the last column of the plate. We cultured the bacteria overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R). In order to have fresh cultures, on the day of sorting, we inoculated 100 μl of the O/N cultures in 900 μl of LB supplemented with ampicillin and tetracycline in deep well plates (VWR) and incubated for 3 h at 37 °C with shaking. We added isopropylthio-β-galactoside (IPTG, 1 mM, Sigma-Aldrich) to the wells to induce the expression of the plasmid for 2 h at 37 °C with shaking. Then, we harvested the cultures by centrifugation (10 min, 3200 g, 4 °C, Eppendorf 5810R) and re-suspended the pellets in 250 μl of S-medium supplemented with carbenicillin (25 μg/ml, Sigma-Aldrich), IPTG (1 mM, Sigma-Aldrich) and cholesterol (5 μg/ml, Sigma-Aldrich).
10
Antibiotic-Mediated Gut Microbiome Depletion
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Endogenous gut microbiomes were depleted using an antibiotic cocktail containing metronidazole (10 mg/ml, Sigma-Aldrich), vancomycin (10 mg/ml, Sigma-Aldrich), neomycin (20 mg/ml, Sigma-Aldrich) and ampicillin (20 mg/ml, VWR, Radnor, PA) in sterile water. The antibiotic cocktail (100 µl) was administered via oral gavage every day for 3 days and then every other day until the end of the experiment. Controls received sterile water alone. Chow was removed from all cages 4 h prior to antibiotic or water gavage to optimize antibiotic absorption. Microbiome depletion was verified using the LIVE/DEAD® BacLight™ Bacterial Viability and Counting Kit (Life Technologies) and subsequent acquisition on the LSRFortessa flow cytometer (BD). For all experiments using antibiotics, mice were housed by group to eliminate cross-contamination from fecal ingestion, provided with autoclaved drinking water and irradiated chow throughout the experiment, and provided with clean autoclaved cages after each antibiotic treatment.
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