The recombinant plasmids pK7WGIGW2R containing RNAi fragments of AmOSCs and an empty vector pK7WG2R-EV as negative control were transformed into Agrobacterium rhizogenes A4 by electroporation respectively. Positive transformants were cultured in LB medium supplemented with spectinomycin (50 mg/L) and kanamycin (50 mg/L) at 28 °C until OD600 = 0.6. Ten milliliters of the bacteria were collected and resuspended using 2 mL of sterilized water containing 50 µM acetosyrengone. Two-week-old A. membranaceus aseptic seedlings were infected with the bacterial solution. The infected seedlings were co-cultured on B5 solid media for one week and transferred onto B5 solid media containing 3% sucrose and 500 mg/L cefotaxime (Sangon Biotech, CN) for about 4 weeks at 25 °C (12 h light/12 h dark). Positive hairy roots were screened out according to the fluorescence of dsRed protein (554 nm for exciting and 586 nm for emitting light), and were then transferred onto a new B5 solid media containing 3% sucrose and 500 mg/L cefotaxime at 25 °C (24 h dark). After 5 weeks, the positive hairy roots were harvested.
Cefotaxime
Manufactured by Sangon
Sourced in China
Cefotaxime is a third-generation cephalosporin antibiotic. It is a white to pale yellow crystalline powder that is soluble in water and in methanol.
Automatically generated - may contain errors
Lab products found in correlation
Erythromycin, by Sangon (2 mentions)
Chloramphenicol, by Sangon (2 mentions)
Trimethoprim, by Sangon (1 mentions)
Tetracycline, by Sangon (1 mentions)
Ofloxacin, by Sangon (1 mentions)
Ampicillin, by Sangon (1 mentions)
Vancomycin, by Sangon (1 mentions)
Penicillin, by Sangon (1 mentions)
Kanamycin, by Sangon (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
Imipenem, by MedChemExpress (1 mentions)
Carbenicillin, by Sangon (1 mentions)
Cellulose nitrate membrane, by Sartorius (1 mentions)
Carboxin, by Merck Group (1 mentions)
Microplate reader, by Bio-Rad (1 mentions)
Meropenem, by Yuanye Bio-Technology (1 mentions)
Ciprofloxacin, by Sangon (1 mentions)
Amikacin, by Sangon (1 mentions)
Acetonitrile and methanol, by Merck Group (1 mentions)
Milli q reagent water system, by Merck Group (1 mentions)
6 protocols using cefotaxime
1
Hairy Root Transformation of Astragalus membranaceus
2
Antibiotic Resistance in S. epidermidis
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Referred study and our pre-experiments show that S. epidermidis can be isolated from the human intestine tract and colostrum, and drug resistance is high [23 (link)]. S. epidermidis was selected for traceability of ARGs because of its high abundance, isolation and resistance rate in the samples. S. epidermidis from different sources was tested for resistance to 10 antibiotics using the K-B method or by determining the minimal inhibitory concentration according to the standards issued by CLSI in 2012 (https://clsi.org/ ). The 10 antibiotics were ampicillin, cefotaxime, penicillin, tetracycline, erythromycin, kanamycin, vancomycin, ofloxacin, chloramphenicol and trimethoprim (Sangon Biotech, Shanghai, China).
3
Bacterial Cultivation and Antibiotic Use
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This study complies with all relevant ethical regulations and the bacterial strains and plasmids used in this study are listed in Table S2 . Unless otherwise noted, S. aureus strains were grown in tryptic soy broth (TSB, Difco) at 37 °C with shaking (250 rpm), or on tryptic soy agar (TSA, Difco) at 37 °C. Escherichia coli strains were grown in Luria-Bertani (LB) broth at 37 °C with shaking (250 rpm) or on LB agar plates at 37 °C. For plasmid maintenance, antibiotics were used at the following concentrations where appropriate: for S. aureus, erythromycin (Sangon Biotech) at 10 μg/ml for RN4220 and 80 μg/ml for USA300 LAC and its derivatives, chloramphenicol (Sangon Biotech) at 15 μg/ml; for E. coli, carbenicillin (Sangon Biotech) at 150 μg/ml. For other reagents, tunicamycin and vancomycin (Dalian Meilun Biotech Co., Ltd.), targocil (Shanghai TopScience Co, Ltd.), oxacillin (Shanghai Aladdin Biochemical Technology Co.,Ltd.), imipenem, cefuroxime, ceftizoxime, cefaclor, cefoxitin, and epicatechin gallate from MedChemExpress, cefotaxime, isopropyl β-D-1-thiogalactopyranoside (IPTG), and AIP from Sangon Biotech, anhydrotetracycline (aTc) from APExBIO, Tarocin A1 from Merck, moenomycin complex from GlpBio, and nile red from Yeasen Biotechnology Co., Ltd. were used in this study.
4
Genetic Transformation of Aspergillus flavus
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A. tumefaciens-mediated transformation of A. flavus was performed as described previously (Han et al. 2018 (link)), with some modification. The A. tumefaciens AGL-1 strain containing the pFC-eGFP plasmid was grown in 10 ml DYT medium supplemented with rifampicin (20 mg/ml) and kanamycin (50 μg/ml) overnight at 28° with shaking at 200 rpm. The overnight culture was then diluted to an OD600 of 0.15 with induction medium (IM) and grown at 28° with shaking at 200 rpm until reaching an OD600 0.35-0.4. The A. tumefaciens cultures were then mixed with an equal volume of A. flavus conidial suspensions (2×106 spores/ml), and subsequently 200 μl of the mixed cultures were plated onto cellulose nitrate membranes (0.45 μm pore, Sartorius Biotech, Goettingen, Germany) placed on co-cultivation medium and grown at 22° for 48 h. The cellulose nitrate membranes were then transferred to selection medium containing 300 μg/ml cefotaxime (Sangon Biotech, China), 60 μg/ml streptomycin (Bomei, China) and 50 μg/ml carboxin (45371, Sigma-Aldrich, Germany) and incubated at 28° in the dark until colonies appeared. The individual colonies were transferred to selection medium with the appropriate antibiotics, as described above, and grown at 28° for 3-4 days.
5
Assessing Sub-MICs of Antibiotics
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Antibiotic sub-MICs were selected by creating a growth curve. K. pneumoniae SW1780 was grown in the presence of 0 × MIC, 1/2 × MIC, 1/4 × MIC, 1/8 × MIC, 1/16 × MIC, 1/ 32 × MIC, 1/64 × MIC, 1/128 × MIC, 1/256 × MIC, 1/512 × MIC, 1/1024 × MIC, 1/2048 × MIC of meropenem (YuanYeBio-Technology, Shanghai, China), ciprofloxacin (Sango Biotech, Shanghai, China), cefotaxime (Sango Biotech, Shanghai, China) and amikacin (Sango Biotech, Shanghai, China). Samples were collected at one-hour intervals and the optical density was read at 595 nm (OD595) using a microplate reader (Bio-Rad, United States). The logarithmic growth rate of the antibiotic stress group and the non-antibiotic group was calculated by applying the logarithmic growth rate formula: U = [ln(Nt) -ln(N0)]/(t-t0), where Nt is the logarithmic growth late OD595 value, N0 is the logarithmic growth early OD595 value, and t-t0 is the logarithmic period time. One-way ANOVA was used to determine statistical differences. The highest antibiotic concentration that did not affect the growth of strain SW1780 was used as sub-MIC.
6
Extraction and Characterization of Bioactive Compounds
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Methyl jasmonate (95%), apigenin 7-O-glucuronide, scutellarin, apigenin, scutellarein, apigenin 7-O-glucoside, naringenin chalcone, naringenin (98%), and acetosyringone were all purchased from Sigma (United States). The antibiotics cefotaxime and hygromycin B were purchased from Sangon (China). The plant hormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (6-BA) were purchased from Phytechnology Laboratories (United States). Ammonium acetate was purchased from Thermo Fisher Scientific (United States) and methanoic acid from Aladdin (China). Methanol and acetonitrile of high performance liquid chromatography (HPLC) grade were from Merck Company (Germany). A Milli-Q Reagent Water System was used to produce ultrapure water (Millipore, United States).
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