The transformation of the pET28-GM-CSF plasmid into E. coli BL21 (DE3), Origami™ 2 (DE3), and SHuffle® T7 cells were performed using heat shock method and positive colonies were selected on Luria-Bertani (LB) plates with antibiotics. A single positive colony from each strain was inoculated in 5 mL of LB broth containing appropriate antibiotics (25 μg/mL tetracycline (Sigma, Germany) + 34 μg/mL kanamycin (Sigma, Germany) for Origami™ 2 (DE3), 34 μg/mL kanamycin + 30 μg/mL spectinomycin (Sigma, Germany) for SHuffle® T7, 34 μg/mL kanamycin for BL21 (DE3) and grown overnight. The next day, this culture was used to inoculate (10% v/v) 25 mL of fresh LB broth in 100 mL shaker flask and incubated at 37 °C and 180 rpm until mid-log phase (i.e., OD600 reached 0.4 to 0.6). The expression of the protein was induced by the addition of 0.5 or 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and incubated at 37, 30, and 23 °C for further 3, 4.5 and 18 h, respectively (14 (link)). The cultures were centrifuged at 3000 g and 4 °C for 10 min and the pellets were stored at -70 °C for future analysis.
Kanamycin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, France, Spain, China, Israel, India, Canada, Macao, Australia, Sao Tome and Principe, Belgium, Sweden, Poland, Japan, Switzerland, Brazil, Italy, Ireland
Kanamycin is a broad-spectrum antibiotic derived from the bacterium Streptomyces kanamyceticus. It is commonly used as a selective agent in molecular biology and microbiology laboratories for the growth and selection of bacteria that have been genetically modified to express a gene of interest.
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Lab products found in correlation
Ampicillin, by Merck Group (284 mentions)
Chloramphenicol, by Merck Group (211 mentions)
Tetracycline, by Merck Group (110 mentions)
Streptomycin, by Merck Group (105 mentions)
Gentamicin, by Merck Group (92 mentions)
Erythromycin, by Merck Group (87 mentions)
Vancomycin, by Merck Group (66 mentions)
Ciprofloxacin, by Merck Group (40 mentions)
Rifampicin, by Merck Group (37 mentions)
Colistin, by Merck Group (34 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (34 mentions)
Glycerol, by Merck Group (32 mentions)
Tween 80, by Merck Group (31 mentions)
Metronidazole, by Merck Group (30 mentions)
Spectinomycin, by Merck Group (30 mentions)
Carbenicillin, by Merck Group (29 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (27 mentions)
Hygromycin, by Merck Group (25 mentions)
Imidazole, by Merck Group (25 mentions)
Gentamycin, by Merck Group (24 mentions)
1 115 protocols using kanamycin
1
Recombinant GM-CSF protein expression
2
Quantifying Engineered Bacteria in Feces and Tumors
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Fecal samples were collected for 18 days after EcN/pMUT-gfp Knr injection. At different time points, about 8–12 feces (~100 mg) were mechanically homogenized in 1 mL sterile PBS. The fecal suspensions were then plated on LB agar plates supplemented with 30 µg/mL kanamycin (Sigma-Aldrich, Schnelldorf, Germany, B5264) and analyzed for EcN/pMUT-gfp Knr presence.
To determine bacterial load in tumor tissues, mice were euthanized and the tumors were excised, weighed and homogenized in sterile PBS. The homogenates were serially diluted and plated on LB agar plates containing 30 µg/mL kanamycin (Sigma-Aldrich, Schnelldorf, Germany, B5264). Resultant colonies were counted and the bacterial numbers were calculated as CFU per 1 g tumor tissue.
To determine bacterial load in tumor tissues, mice were euthanized and the tumors were excised, weighed and homogenized in sterile PBS. The homogenates were serially diluted and plated on LB agar plates containing 30 µg/mL kanamycin (Sigma-Aldrich, Schnelldorf, Germany, B5264). Resultant colonies were counted and the bacterial numbers were calculated as CFU per 1 g tumor tissue.
3
Genetic Manipulation of MRSA Strain JE2
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The strains and plasmids used in this study are listed in Table S1 . The methicillin-resistant S. aureus (MRSA) USA300 strain JE2 (GenBank CP000255 ) was used throughout the study. The deletion mutant of hflX (SAUSA300_1198) has been described previously (31 (link)). Unless otherwise noted, S. aureus cells were grown at 37 °C in tryptic soy broth (TSB, Difco) at a 5:1 tube- or flask-to-medium ratio with a 1:100 dilution of an overnight seed culture. TSB agar plates were prepared using DifcoTM agar (Difco, BD 281230), Eiken agar (Eiken, E-MJ00), Gelrite (RPI, G35020), and agarose (GenMate, E-3120). When necessary, erythromycin, chloramphenicol, cadmium chloride, kanamycin, and anhydrotetracycline (all from Sigma-Aldrich) were used at 5 μg/ml, 10 μg/ml, 0.15 mm , 75 μg/ml, and 400 ng/ml, respectively. The restriction-deficient S. aureus RN4220 strain was used as a plasmid passage surrogate before the plasmid was transformed into the destination JE2 derivatives. E. coli cells harboring expression vectors were grown at 16 or 37 °C in LB (Difco). Antibiotics were used at 50 (kanamycin) or 100 μg/ml (ampicillin, Sigma-Aldrich). All GTP analogs were from Sigma-Aldrich. Primers were purchased from IDT DNA and are listed in Table S2 .
4
Survival of L. paracasei DTA81 in GIT
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The survival of L. paracasei DTA81 after transit through the GIT was evaluated at the end of week 3 and at the end of the study (end of week 6). Three mice from each group (from different cages) were randomly selected and their faeces were collected, weighed resuspended in 10 ml of sterilized PBS and serially diluted using the same solution. Then they were plated on MRS medium supplemented with kanamycin (64 μg ml−1; Sigma) and incubated at 37°C for 48 h. Resistance of L. paracasei DTA81 to kanamycin had been determined in a previous study (Tarrah et al. 2019 ). After incubation, colony forming units were counted and reported per gram of wet faeces. Besides, five colonies were also randomly taken from plates and investigated by Gram staining, catalase and oxidase tests. The same mice were used for this evaluation at week 3 and at week 6.
5
Cultivation and Maintenance of Vibrio vulnificus and Escherichia coli
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All V. vulnificus strains were grown in heart infusion broth (Difco) supplemented to 2% NaCl (HI) and on HI agar plates containing 18 g/l of agar (Difco). Broth cultures were incubated at 30°C and 200 rpm; plates were incubated overnight (ON) for 16–24 h at 30°C. Phase switching assays in HI and growth curves were all performed as previously described [8] (link). Escherichia coli strains were grown in LB broth (Difco), broth cultures were incubated at 37°C and 250 rpm, and plates were incubated ON for 16–24 h at 37°C. Antibiotics (Sigma) were used at the following concentrations: 150 µg/ml kanamycin, 50 µg/ml ampicillin, and 2 µg/ml chloramphenicol for V. vulnificus and 50 µg/ml kanamycin, 50 µg/ml ampicillin, and 10 µg/ml chloramphenicol for E. coli. Arabinose (Sigma) was typically added to a final concentration of 0.2% when needed. E. coli and V. vulnificus strains used or created in this study are listed in Table 1 .
6
Proteus mirabilis HI4320 Transposon Mutant Library
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Proteus mirabilis HI4320 was isolated in a prior study from the urine of a catheterized patient in a chronic care facility in Baltimore, Maryland [2 (link), 59 (link)]. The P. mirabilis HI4320 transposon mutant library was previously constructed, validated, and successfully utilized in a mouse model of CAUTI [28 (link)]. Bacteria were routinely cultured at 37°C with aeration in 5 ml LB broth (10 g/L tryptone, 5 g/L yeast extract, 0.5 g/L NaCl) or on LB solidified with 1.5% agar. Proteus mirabilis minimal salts medium (PMSM) (10.5 g/L K2HPO4, 4.5 g/L KH2PO4, 0.47 g/L sodium citrate, 1 g/L (NH4)2SO4, supplemented with 0.001% nicotinic acid, 1mM MgSO4, and 0.2% glycerol) [60 (link)] or RPMI with 2 mM L-glutamine (Sigma). Transposon mutants were cultured in LB containing 25 μg/ml kanamycin (Sigma). Additional P. mirabilis mutants for validation of candidate fitness factors were constructed by insertion of a kanamycin resistance cassette as previously described using the TargeTron system (Sigma), and are listed in S8 Table [61 (link)]. Resulting mutants were screened by kanamycin selection and PCR. All primers for generation and verification of mutants are provided in S9 Table .
7
Bacterial Growth Optimization with Antibiotics
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Bacterial strains and plasmids used in this study can be found in Table 1 . Bacteria were grown in lysogeny broth (LB) or on LB agar at 37°C for E. coli and at 30°C for B. thuringiensis unless stated otherwise. When necessary, media were supplemented with antibiotics (Sigma-Aldrich, Overijse, Belgium), i.e., 50 μg·mL−1 kanamycin (pET30a selection), 100 μg·mL−1 ampicillin (pHT304pxyl or pHT1618Kpxyl selection in E. coli ), 200 μg·mL−1 kanamycin (pHT1618Kpxyl selection in B. thuringiensis ), 10 μg·mL−1 erythromycin (pHT304pxyl selection in B. thuringiensis ), or 10 μg·mL−1 chloramphenicol (E. coli Rosetta growth).
8
Isolation and Genomic Analysis of Bacillus velezensis Protease Producers
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Four FE products in solid form were collected from the market (Table 1 ). One gram of each batch tested from each FE product was added to 250 mL of Brain–Heart Infusion broth (Sigma-Aldrich) in the presence of Kanamycin (50 µg/mL; Sigma-Aldrich) for incubation overnight at 30 °C. An amount of 100 μL of the culture was plated on nutrient agar (Sigma-Aldrich) in the presence of Kanamycin (50 µg/mL; Sigma-Aldrich) for incubation overnight at 30 °C. Based on real-time PCR analysis (see Section 2.2 ), isolates of the GM B. velezensis producing protease were selected for genomic analysis. For each batch tested from each FE product, two isolates were selected, to investigate the presence of potentially different strains, as well as to serve as a back-up against potential loss of plasmids from the isolates.
9
E. coli Nissle 1917 and K12 MG1655 Cultures
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E. coli Nissle 1917 wild-type (EcN WT) (Ardeypharm GmbH, Herdecke, Germany) and E. coli K12 MG1655 wild-type (CGSC7740) were used in this study. E. coli BW29427, diamonopimelic acid auxotroph and:: pir (Datsenko KA and Wanner BL, Purdue University) served as the mini-Tn5 donor strain. All strains were grown in Luria-Bertani (LB) (Fisher Scientific, UK) at 37°C with shaking. Media were supplemented with 50 μg ml–1 kanamycin (Km) for culture of mini-Tn5 transposon donor strain, 30 μg ml–1 kanamycin and 100 nM diamonopimelic acid (DAP) (Sigma-Aldrich, UK) for BW29427. Where appropriate the growth media was supplemented with 20 μg ml–1 chloramphenicol (Cm), 12 μg ml–1 tetracycline (Tc), or 200 μg ml–1 gentamicin (Gen) (Sigma, UK).
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E. coli Nissle 1917 wild-type (EcN WT) (Ardeypharm GmbH, Herdecke, Germany) and E. coli K12 MG1655 wild-type (CGSC7740) were used in this study. E. coli BW29427, diamonopimelic acid auxotroph and:: pir (Datsenko KA and Wanner BL, Purdue University) served as the mini-Tn5 donor strain. All strains were grown in Luria-Bertani (LB) (Fisher Scientific, UK) at 37°C with shaking. Media were supplemented with 50 μg ml–1 kanamycin (Km) for culture of mini-Tn5 transposon donor strain, 30 μg ml–1 kanamycin and 100 nM diamonopimelic acid (DAP) (Sigma-Aldrich, UK) for BW29427. Where appropriate the growth media was supplemented with 20 μg ml–1 chloramphenicol (Cm), 12 μg ml–1 tetracycline (Tc), or 200 μg ml–1 gentamicin (Gen) (Sigma, UK).
10
In vitro analysis of M. tuberculosis mutants
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For in vitro experiments, M. tuberculosis H37Rv (WT), pMV (empty plasmid control) and OVER (MTS1338 overexpressing) M. tuberculosis strains were initially grown from frozen stocks for 10 days in Sauton medium. Medium content (per liter): 0.5 g KH2PO4, 1.4 g MgSO4×7H2O, 4 g L-asparagine, 60 ml glycerol, 0.05 g ferric ammonium citrate, 2 g sodium citrate, 0.1 ml 1% ZnSO4, pH 7.0 (adjusted with 1M NaOH). Supplements: ADC growth supplement (Connell, 1994 (link)), 0.05% Tween 80 and 50 μg/ml kanamycin (Sigma-Aldrich, USA). Growth conditions: 37°C with agitation (200 rpm). The starter cultures were inoculated into fresh medium (the same composition) and grown up to stationary phase for RNA-seq experiments and stress survival experiments.
For cloning procedures, Escherichia coli DH5α was grown in Luria Bertani (LB) broth and LB-agar. When required, antibiotics were added at the following concentrations: kanamycin (Sigma-Aldrich), 50 μg/ml (M. tuberculosis); ampicillin (Invitrogen, USA), 100 μg/ml (E. coli).
For cloning procedures, Escherichia coli DH5α was grown in Luria Bertani (LB) broth and LB-agar. When required, antibiotics were added at the following concentrations: kanamycin (Sigma-Aldrich), 50 μg/ml (M. tuberculosis); ampicillin (Invitrogen, USA), 100 μg/ml (E. coli).
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