Ampicillin

Manufactured by Research Products International

Sourced in Israel

Ampicillin is a broad-spectrum antibiotic used in research applications. It inhibits the synthesis of bacterial cell walls, effectively killing or preventing the growth of certain types of bacteria.

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5 protocols using ampicillin

Antibiotic Screening and Growth Curve

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Cultures for growth curve and antibiotic screening procedures were started from seed culture diluted to 0.02 OD₆₀₀ or 2.5% (v/v), respectively, and were grown in 10 g/L TSBG. New tubes were opened at each time point to maintain anaerobic conditions throughout and all measurements were made in biological triplicate. Antibiotics used included kanamycin (Teknova, Hollister, CA), tetracycline (Thermo Scientific, Waltham, MA), ampicillin (Research Products International, Mount Prospect, IL), gentamicin (Fisher Scientific, Waltham, MA), spectinomycin (VWR, Radnor, PA), and chloramphenicol (VWR, Radnor, PA) at concentrations listed in Results and Discussion. Growth repression for each sample was calculated using Eq 1. Each sample OD₆₀₀ (G_S) was divided by average growth in wild type (G_WTavg), converted to a percentage and subtracted from 100 to obtain the percent repression. Growth repression was averaged across three sample replicates for each treatment.

Long D.S., Immethun C.M., Vallecilla-Yepez L., Wilkins M.R, & Saha R. (2021). One step forward, two steps back: Transcriptional advancements and fermentation phenomena in Actinobacillus succinogenes 130Z. PLoS ONE, 16(5), e0245407.

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Purification and Analysis of Proteins

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Restriction nucleases, ligases and competent cells were from New England Biolabs, plasmid purification kits from Promega and restriction digest clean-up kits from Qiagen. Ni Sepharose 6 Fast Flow was from General Electric. Salts, 2-mercaptoethanol, 37% (v/v) formaldehyde, imidazole, DTT, THF, Tris and acetonitrile were from Sigma. CHAPS was from Thermo Fisher Scientific. LB (Luria–Bertani) broth extract was from Becton, Dickinson and Company. IPTG (isopropyl β-D-thiogalactopyranoside) and ampicillin were from Research Products International. 5-FU was from MP Biomedicals. Amicon Ultra-15 Centrifugal Filter Units were from Millipore. All experiments were conducted in ultra-pure RNase-free water.

Brunn N.D., Dibrov S.M., Kao M.B., Ghassemian M, & Hermann T. (2014). Analysis of mRNA recognition by human thymidylate synthase. Bioscience Reports, 34(6), e00168.

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Purification and Characterization of Rhizobium etli PC

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IPTG, D-biotin, ampicillin, kanamycin, chloramphenicol, dithiothreitol (DTT), Tris buffer, epoxysuccinyl-L-leucylamido(4-guanido) butane (E-64), and pepstatin A were purchased from Research Products International Corp (Mount Prospect, IL). Lactate dehydrogenase was purchased from Roche Diagnostics (Indianapolis, IN). Acetyl-CoA was purchased from Crystal Chem, Inc (Downers Grove, IL). Granulated LB broth (Miller's modification) was purchased from EMD Millipore Chemicals, Inc. All other materials were purchased from Sigma-Aldrich. PC from Rhizobium etli (RePC) was previously subcloned into a modified pET-17b vector [20 (link)]. ΔBC RePC [23 (link)] and ΔBCΔBCCP RePC [11 (link)] were previously subcloned into a modified pET-28a vector.

Lietzan A.D., Lin Y, & St. Maurice M. (2014). The role of biotin and oxamate in the carboxyl transferase reaction of pyruvate carboxylase. Archives of biochemistry and biophysics, 562, 70-79.

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Antibiotic Susceptibility Profiling of βLR16 Clones

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The antibiotic susceptibility of βLR16 and subclones was tested in minimum inhibitory concentration assays according to CLSI guidelines except for lower incubation temperatures (24–28°C) [20 (link)]. Briefly, serial two-fold dilutions (from 512 μg ml^-1 to 0.5 μg ml^-1, no antibiotic in the last well) of antibiotics were made in Mueller-Hinton (MH) broth (Becton, Dickinson and Company, Sparks, MD, USA) in 96-well plates. The following antibiotics were tested: ampicillin, rifampin (both from Research Products International Corp., Mt. Prospect, IL, USA), carbenicillin (Fisher Scientific, Fair Lawn, NJ, USA), ciprofloxacin (Wako Chemicals USA, Inc., Richmond, VA), erythromycin (Fluka BioChemika, Buchs, Switzerland), amoxicillin, cefamandole, cefoxitin, ceftazidime, cephalexin, piperacillin, chloramphenicol, nalidixic acid, fusidic acid, and gentamicin (all from Sigma, St. Louis, MO, USA). Each well was inoculated with 10 μl containing 10⁵ colony forming units of each clone being tested. The assay was performed in duplicate at least three times, and E. coli strains EPI300, BL21(DE3), or BW25113 (Table 1) containing empty vector always served as the negative controls. The minimum inhibitory concentration (MIC) corresponded to the antibiotic concentration of the first well in which no growth was visible.

Allen H.K., An R., Handelsman J, & Moe L.A. (2015). A Response Regulator from a Soil Metagenome Enhances Resistance to the β-Lactam Antibiotic Carbenicillin in Escherichia coli. PLoS ONE, 10(3), e0120094.

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Ras Protein Purification from E. coli

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E. coli BL21 cells were obtained from New England Biolabs (Ipswich, MA), and Bacto yeast extract, agar and tryptone for bacterial growth were from Gibco (Waltham, MA). Ampicillin and HEPES free acid were from Research Products International (Mount Prospect, IL). Kanamycin and reduced L-glutathione were from Sigma-Aldrich (St. Louis, MO). Phenylmethylsulfonyl fluoride (PMSF) used to inhibit proteolysis during Ras purification was from Roche Diagnostics (Basel, Switzerland). TALON cobalt(II) metal affinity resin for polyhistidine tag purification was from Takara (Kusatsu Japan), and HYDRANAL imidazole was from Honeywell (Charlotte, NC). Ethylenediamine tetracetic acid disodium salt dihydrate (EDTA) was from Thermo Fisher Scientific (Waltham, MA). Non-hydrolyzable GTP analog GMPPNP, conjugated with four lithium counter ions, at >95% purity, was from Abcam (Cambridge, UK). Guanosine 5’-diphosphate [GDP] disodium salt was from Sigma-Aldrich (St. Louis, MO). Desalting columns were Econo-Pac 10DG desalting prepacked gravity flow columns from Bio-Rad (Hercules, CA). Vivaspin 500, 10,000 MWCO spin concentrators from Sartorius were used to concentrate protein samples (Göttingen, Germany). Microscale thermophoresis measurements were performed on a Nanotemper Monolith NT.115 instrument utilizing Monolith NT.115 capillaries (Munich, Germany).

Fleming I., Hannan J.P., Swisher G.(., Tesdahl C.D., Martyr J.G., Cordaro N.J., Erbse A.H, & Falke J.J. (2022). Binding of Active Ras and Its Mutants to the Ras Binding Domain of PI-3-Kinase: A Quantitative Approach to KD Measurements. Analytical biochemistry, 663, 115019.

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