C. crescentus strains were grown in minimal (M2G) or nutrient-rich media (PYE) from a freezer stock inoculum, at 30°C and 180 rpm. Liquid media was supplemented with 5 μg/mL Kanamycin (Kanamycin sulfate in water, IBI Scientific), 0.2 μg/mL Chloramphenicol (in 100% ethanol, Sigma Aldrich), 0.4 μg/mL Gentamycin (Gentamycin sulfate in water, Sigma Aldrich), 1.25 μg/mL Spectinomycin (Sigma Aldrich). PYE plates contained 25 μg/mL Kanamycin or 1 μg/mL Chloramphenicol. C. crescentus cells were synchronized using the mini-synchrony protocol to isolate a nascent population of swarmer cells (37 ). In short, cells were grown in M2G (15 mL) to OD600 ~0.3 and pelleted by spinning at 6000 rpm for 10 min at 4°C. The cell pellet was resuspended in 800 μL of 1 × M2 salts, and then mixed with 900 mL Percoll (Sigma-Aldrich). The mixed solution was then transferred to a 2 mL microfuge tube and centrifuged at 11,000 rpm for 20 min at 4°C to separate the swarmer from stalked cells via a density gradient. The swarmer cells were extracted by collecting the bottom layer of the gradient, placed in another tube to be washed twice with 1 × M2 salts, centrifuging at 8000 rpm for 3 min at 4°C. The swarmer cells were resuspended in M2G medium (2 mL, OD600 ~0.1). PYE medium was used during C. crescentus strain constructions.
Kanamycin
Manufactured by IBI Scientific
Sourced in United States
Kanamycin is an antibiotic used for selective growth of bacterial cultures in laboratory settings. It inhibits bacterial protein synthesis, making it effective against a wide range of gram-positive and gram-negative bacteria.
Automatically generated - may contain errors
Lab products found in correlation
Tetracycline, by Thermo Fisher Scientific (2 mentions)
Co no3 2 6h2o, by Merck Group (2 mentions)
H3bo3, by Thermo Fisher Scientific (2 mentions)
Synergy h1 hybrid plate reader, by Agilent Technologies (2 mentions)
Cuso4 5h2o, by Merck Group (2 mentions)
Chloramphenicol, by Thermo Fisher Scientific (2 mentions)
Rifampicin, by Merck Group (1 mentions)
Znso4 7h2o, by Merck Group (1 mentions)
Luria broth, by Thermo Fisher Scientific (1 mentions)
Na2moo4 2h2o, by Thermo Fisher Scientific (1 mentions)
Phosphocreatine, by Merck Group (1 mentions)
Acetoin, by Merck Group (1 mentions)
Gentamycin, by Teknova (1 mentions)
Phenylboronic acid, by Merck Group (1 mentions)
Spectinomycin, by MP Biomedicals (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
U 13c glucose, by Cambridge Isotopes (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
7 protocols using kanamycin
1
Synchronizing Caulobacter crescentus Cells
2
Bacterial Infection Assay Protocol
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P. syringae pv. tomato DC3000 (Pst) and Pst avrRpm1 (Psta) were used for bacterial infection assays and ETI elicitations. A single colony of Pst was grown in 2 ml of LB medium containing 25 μg/ml rifampicin (Sigma-Aldrich). A single colony of Psta from a freshly streaked 3-day-old agar plate was grown in 50 ml of LB medium containing 25 μg/ml rifampicin and 50 μg/ml kanamycin (IBI Scientific, Peosta, IA). Both strains were grown to log phase, washed in sterile water twice or once, respectively, resuspended in sterile water to OD600 of 0.2, and incubated at room temperature with no agitation for 6 and ~2.5 h, respectively, prior to infection. Bacterial infection assays on 4- to 5-week-old adult leaves were performed as described in Barco et al. (2019b) (link).
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P. syringae pv. tomato DC3000 (Pst) and Pst avrRpm1 (Psta) were used for bacterial infection assays and ETI elicitations. A single colony of Pst was grown in 2 ml of LB medium containing 25 μg/ml rifampicin (Sigma-Aldrich). A single colony of Psta from a freshly streaked 3-day-old agar plate was grown in 50 ml of LB medium containing 25 μg/ml rifampicin and 50 μg/ml kanamycin (IBI Scientific, Peosta, IA). Both strains were grown to log phase, washed in sterile water twice or once, respectively, resuspended in sterile water to OD600 of 0.2, and incubated at room temperature with no agitation for 6 and ~2.5 h, respectively, prior to infection. Bacterial infection assays on 4- to 5-week-old adult leaves were performed as described in Barco et al. (2019b) (link).
3
Optimized Production of Bacterial Esters
4
Metabolic Pathway Analysis with Isotopic Labeling
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The following reagents were obtained from Sigma-Aldrich: glucose, 1,3-propanediol, phenylboronic acid, cycloheximide, 23BD, acetoin, pyruvate, PEP, 6PG, G6P, F6P, Ru5P, R15P, ATP, dithiothreitol (DTT), NADH, NADPH, NADP+, phosphocreatine, RuBisCO, pyruvate kinase, lactate dehydrogenase, G6P dehydrogenase, 3PGA kinase, GAPDH and creatine kinase. U-13C glucose and 13C-NaHCO3 were obtained from Cambridge Isotope Laboratories. IPTG and chloramphenicol were obtained from Fischer Scientific. Gentamycin was purchased from Teknova. Spectinomycin was purchased from MP Biomedicals. Kanamycin was purchased from IBI Scientific. Phusion polymerase was purchased from New England Biolabs. All oligonucleotide synthesis and DNA sequencing were performed by Eurofins MWG Operon Inc.
5
Bacterial Culturing Conditions for M. marinum and E. coli
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Mycobacterium marinum M bacterial strains were grown on Middlebrook’s 7H11 (Sigma-Aldrich) agar plates supplemented with 10% OADC [oleic acid (Fischer Scientific), dextrose (Sigma and VWR), albumin (Sigma Aldrich), and catalase(Fischer)] and 0.5% glycerol (VWR, Radnor, PA) at 30°C or in Middlebrook’s 7H9 media (Sigma-Aldrich, St. Louis, MO) supplemented with 10% OADC and 0.2% tyloxapol (Chem-Impex Int’l Inc., Wood Dale, IL) at 30°C. Hygromycin (EMP Millipore) and Kanamycin (IBI Scientific, Peosta, IA) were added to final concentrations of 50 μg/mL and 20 μg/mL, respectively, where noted. Escherichia coli was grown on Luria-Bertani (LB) agar or in LB broth at 37°C. Kanamycin, Hygromycin, and Ampicillin (VWR) were added to final concentrations of 50 μg/mL, 200 μg/mL, and 200 μg/mL when stated. Please see S1 Table for a complete list of bacterial strains used in this study.
6
Optimized Bacterial Culture and IBA Production
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Overnight cultures were grown in 5 ml Luria broth media containing appropriate antibiotics. Antibiotic concentrations were as follows: kanamycin (50 μg ml−1; IBI Scientific), ampicillin (200 μg ml−1; Fisher BioReagents), tetracycline (20 μg ml−1; Fisher BioReagents) and spectomycin (50 μg ml−1). IBA production was carried out with M9P medium, consisting of M9 medium (33.7 mM Na2HPO4, 22 mM KH2PO4, 8.55 mM NaCl, 9.35 mM NH4Cl, 1 mM MgSO4 and 0.1 mM CaCl2; BD Bacto); 5 g l−1 yeast extract (BD Bacto); 50 g l−1 glucose (Fisher BioReagents); and 1,000-fold dilution of A5 trace metal mix (2.86 g H3BO3 (Fisher Chemical), 1.81 g MnCl2·4H2O (Alfa Aesar), 0.079 g CuSO4·5H2O (Sigma-Aldrich) and 49.4 mg Co(NO3)2·6H2O (Sigma-Aldrich) per liter water). Optical densities (OD) were measured at 600 nm with a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).
7
Bacterial Growth Media Preparation
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All media components were purchased from Fisher Scientific (Waltham, MA, USA) unless otherwise noted. Lysogeny broth (LB) was prepared by adding 10 g of tryptone powder, 10 g of sodium chloride, and 5 g of yeast per liter of deionized water. All LB media were adjusted to a pH of 7.5. The LB plates and top agar were made by adding 17 g and 6 g, respectively, of agar to 1 L of the LB media. When used, kanamycin (IBI Scientific, Las Vegas, NV, USA) was added to the LB plates at a final concentration of 50 µg/mL. Modified tryptone soya broth (mTSB) was made according to the USDA-FSIS Media and Reagents guideline (USDA-FSIS 2022).
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