E. coli C41 (DE3) cells (Sigma-Aldrich) were transformed with the plasmids pNR31 and pNR33. 100 mL Luria-Bertani (Fisher Scientific) liquid cultures (100 μg/mL ampicillin, Sigma-Aldrich) were inoculated 1:100 from overnight cultures. The E. coli cells were grown at 37∘C while shaking at 220 rpm until an OD600 of 0.4 was reached. Then, all-trans-retinal (Sigma-Aldrich) was added to a concentration of 10 μM and the expression of the fusion-proteins was induced with the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG, Sigma-Aldrich). The cells were incubated for another 4 h at 37 ∘C while shaking at 220 rpm. Subsequently they were harvested by centrifugation (3200 × g for 10 min at 4 ∘C) and resuspended in 150 mM NaCl. The cells were stored at 4∘C and protected from light until further use.
Luria bertani
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
Luria-Bertani (LB) is a commonly used nutrient-rich growth medium for culturing bacteria. It provides the essential nutrients, vitamins, and minerals required for bacterial growth and proliferation. LB is a versatile medium that can be used for a wide range of bacterial species.
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Lab products found in correlation
Ampicillin, by Merck Group (2 mentions)
M17 broth, by Thermo Fisher Scientific (2 mentions)
Isopropyl d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Nutrient agar, by Thermo Fisher Scientific (1 mentions)
Brain heart infusion broth, by Thermo Fisher Scientific (1 mentions)
Listeria innocua atcc 33090, by American Type Culture Collection (1 mentions)
M9 minimal media, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Vitox, by Thermo Fisher Scientific (1 mentions)
E z n a plasmid mini kit 1, by Omega Bio-Tek (1 mentions)
Plasmid mega kit, by Qiagen (1 mentions)
Macconkey agar, by Thermo Fisher Scientific (1 mentions)
Chitosan, by Merck Group (1 mentions)
Lm17 broth, by Thermo Fisher Scientific (1 mentions)
Tryptone, by Thermo Fisher Scientific (1 mentions)
Yeast extract, by Thermo Fisher Scientific (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Gentamicin gen, by Merck Group (1 mentions)
33 protocols using luria bertani
1
Expression of Retinal-Binding Proteins in E. coli
2
Evaluation of Biofilm Dispersal Agents
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The pathogenic E. coli strains were isolated from outbreaks attributed to vegetables: E. coli O157:H7 LJH0537, E. coli O157:H7 LJH1186, E. coli O157:H7 LJH643, E. coli O145 RM12333 (Selma et al. 2008 (link)). Salmonella enterica (isolated from vegetables outbreaks): S. enterica serovar Typhimurium ATCC14028, sv. Braenderup 04E01347, Braenderup 04E01556, Braenderup 04E00783, sv. Montevideo LJH519, sv. Javiana ATCC BAA-1593 and sv. Newport C6.3 (Noel et al. 2010 ). Listeria innocua ATCC33090 was purchased from ATCC (Teddington, Middlesex, UK). We were also interested in testing the effect of nitric oxide donors on dispersing biofilm formed by plant pathogens; It is well know that they can form biofilm in irrigation pipes (Narayanasamy 2011 ; Hong et al. 2014 ). The following plant pathogens were used: Pectobacterium carotovorum SR38, and Xanthomonas oryzae pv.oryzae J18. All strains were maintained as frozen glycerol stocks, and were sub-cultured into Luria–Bertani (Fisher, Waltham, MA, USA), Nutrient Agar (Oxoid, Basingstoke UK) or Brain Heart Infusion broth (Oxoid, Basingstoke UK) media.
3
Cultivation and Maintenance of Salmonella and E. coli
4
Vibrio cholerae Strain Construction
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V. cholerae strains in this study are derivatives of V. cholerae WT El Tor strain N16961 (Heidelberg et al., 2000 (link)). Construction of plasmids and mutant V. cholerae strains is described in the next section along with a table of strains and plasmids used in this work (Supplementary file 4 ).
Strains were grown at 30 or 37°C in Luria-Bertani (LB-Miller, Fisher Bioreagents# BP97235) with or without 1% NaCl, 1% sucrose, or 10% sucrose, or in M9 minimal media + 0.4% glucose (Cold Spring Harbor Protocols) where indicated in the figure legends. Growth media were supplemented with kanamycin (50 μg/mL), ampicillin (25 μg/mL), or chloramphenicol (5 μg/mL) in plates and overnight cultures when needed to maintain plasmids or chromosomal integration of suicide vectors. Genes under Para and Ptac regulation were induced with 0.4% L-arabinose or 200 μM isopropyl-β-D-1-thiolgalactopyranoside (IPTG), respectively.
Strains were grown at 30 or 37°C in Luria-Bertani (LB-Miller, Fisher Bioreagents# BP97235) with or without 1% NaCl, 1% sucrose, or 10% sucrose, or in M9 minimal media + 0.4% glucose (Cold Spring Harbor Protocols) where indicated in the figure legends. Growth media were supplemented with kanamycin (50 μg/mL), ampicillin (25 μg/mL), or chloramphenicol (5 μg/mL) in plates and overnight cultures when needed to maintain plasmids or chromosomal integration of suicide vectors. Genes under Para and Ptac regulation were induced with 0.4% L-arabinose or 200 μM isopropyl-β-D-1-thiolgalactopyranoside (IPTG), respectively.
5
E. coli Proteome Sample Preparation
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E. coli DH5-α was cultured overnight in Luria-Bertani (Thermo Fisher Scientific) broth at 37 °C with shaking at 180 rpm. The next day, cells were harvested (4000 xg, 15 min, 4 °C), washed twice with ice-cold PBS pH 7.4 and stored at −80 °C until further analysis. Cell pellets were lysed in 5% SDS in 50 mM triethylammonium bicarbonate (TEAB) pH 7.5, by sonication. Whole-cell lysates were subsequently centrifuged (10,000 xg, 5 min, RT) and the supernatants used for proteomic sample preparation.
6
NaCl Effects on Bacterial Growth and Viability
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The effect of NaCl on the growth of bacteria was studied on a Luria–Bertani (Thermo Fisher Scientific, Waltham, MA, USA) medium with the addition of various concentrations of NaCl (1%, 5%, 10%, 15%, and 20%) [47 (link)]. The Petri dishes were incubated at a temperature of 28 °C. The growth of bacteria was noted visually after 72 h. The effect of NaCl on the cell number of TS3 was studied in the liquid LB medium supplemented with various concentrations of NaCl (1%, 5%, 10%, 15%, and 20%). The TS3 was cultured for 3 days at 28 °C in 500 mL flasks placed in a rotary shaker (180 rpm). Then, aliquots (100 µL) of the serial dilutions (10−1–10−5) were inoculated onto LB plates. The plates were maintained at 28 °C for 3 days, and the cell number was calculated.
7
Culturing and Anaerobic Shock of Salmonella Typhimurium
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A complete list of bacterial strains used in this study is provided in Supplementary Table S1 . Salmonella enterica serovar Typhimurium strain SL1344 (QGS-001) is referred to as wild type and was used for mutant construction. Bacteria were grown at 37°C with continuous shaking at 220 rpm in Luria-Bertani (Oxoid™) or M9CA minimal medium supplemented with 0.4% glucose and 20 mM sodium nitrate.
Overnight cultures were grown from a single colony, diluted 1:100 in fresh medium, and grown to the indicated OD600. Where appropriate, media were supplemented with antibiotics at the following concentrations: 100 μg/ml ampicillin (Amp), 50 μg/ml kanamycin (Kan) and 20 μg/ml chloramphenicol (Cm). Unless stated otherwise, chemicals were purchased from Sangon Biotech, Shanghai. For anaerobic shock (33 (link)), cells were aerobically cultured to an OD600 of 0.3, filled in 50 ml closed Falcon tubes, and incubated without agitation at 37°C for the indicated time. For heat shock in the rne-TS experiment, bacteria were grown in LB at 28°C to an OD600 of 0.3, and then grown for 30 min at 28°C or 44°C in sealed Falcon tubes (anaerobic shock condition).
Overnight cultures were grown from a single colony, diluted 1:100 in fresh medium, and grown to the indicated OD600. Where appropriate, media were supplemented with antibiotics at the following concentrations: 100 μg/ml ampicillin (Amp), 50 μg/ml kanamycin (Kan) and 20 μg/ml chloramphenicol (Cm). Unless stated otherwise, chemicals were purchased from Sangon Biotech, Shanghai. For anaerobic shock (33 (link)), cells were aerobically cultured to an OD600 of 0.3, filled in 50 ml closed Falcon tubes, and incubated without agitation at 37°C for the indicated time. For heat shock in the rne-TS experiment, bacteria were grown in LB at 28°C to an OD600 of 0.3, and then grown for 30 min at 28°C or 44°C in sealed Falcon tubes (anaerobic shock condition).
8
Bacterial Strains and Biosensors for AHL Detection
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Escherichia coli DH5α was used as the cloning host and E. coli MG1655 was used for proteins expression and AHLs production. E. coli JM109 [pSB536] and E coli JM109 [pSB1075] are lux-based AHL biosensors40 (link)41 (link). P. aeruginosa PAO1 was cultured as AHL production positive control. All bacterial strains were routinely grown on LB (Luria-Bertani, Oxoid Ltd. UK) agar plates and in LB broth with 200 rpm shaking with appropriate antibiotics at 37°C for E. coli and P. aeruginosaPAO1 and 28°C for C. violaceum CV026 biosensor42 (link). Antibiotics were used at the following concentrations: ampicillin, 100 mg/ml; tetracycline, 10 mg/ml (Sigma Aldrich, UK). Growth curve and luminescence was quantified in 96-well microtitre plates by using Tecan luminometer (Infinite M200Pro).
9
Antibiotic Susceptibility Assay Protocol
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Bacterial strains, plasmids, and oligonucleotide primers used in this study are shown in Supplementary Table S1 . All strains were grown at 37°C in Luria-Bertani (LB, Oxoid, United Kingdom) broth unless otherwise stated. Mueller-Hinton (MH, Beijing Land Bridge Technology, China) broth dilution was used to determine the minimal inhibitory concentration (MIC) of antibiotics. Antibiotic disk (Hangzhou Microbial Reagent, China) diffusion tests were used for the antibiotic susceptibility assay (cephalexin and cefazolin). Appropriate antibiotic agents were added when preparing the seed liquid of all bacteria possessing a plasmid. Then, the seed liquid was used directly in relevant experiments.
10
Growth Conditions for Bacterial Strains
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Bacterial strains used in this study are listed in Table 1 . N. meningitidis were grown on brain heart infusion (BHI) agar (1.5% [wt/vol], Oxoid) supplemented with 5% Levinthal's base (500 mL defibrinated horse blood autoclaved with 1 L BHI). N. gonorrhoeae was grown in a gonococcal base liquid medium (GCBL) consisting of 1.5% (wt/vol) proteose peptone number 3 (Becton, Dickinson), 0.1% (wt/vol) starch, 0.4% (wt/vol) K2HPO4, 0.1% (wt/vol) KH2PO4, 0.5% (wt/vol) NaCl supplemented with 1% Vitox (Oxoid). Agar 1.5% (wt/vol) (Oxoid) was included for solid medium. Cultures were then incubated for 16 to 18 h at 37°C with 5% CO2. Escherichia coli was grown on Luria-Bertani agar (Oxoid) overnight at 37°C. For growth assays, measurements of the Optical Density (A600) were taken every hour for 4 h. N. gonorrhoeae was sialylated by the addition of 2 μg mL−1 of cytidine-5′-monophosphate-N-acetylneuraminic acid (CMP-NANA) to media. For Neisseria spp., kanamycin (kan) and erythromycin were added to media at 50 μg mL−1 and 2 μg mL−1, respectively. Ampicillin (100 μg mL−1) was used for Escherichia coli as required.
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