The antibiotics gentamycin, rifampicin, and kanamycin were obtained from Serva (Heidelberg, Germany); ampicillin was purchased from Carl Roth GmbH (Karlsruhe, Germany). Anhydrotetracycline hydrochloride, desthiobiotin, and Strep-Tactin Superflow chromatography material were obtained from IBA GmbH (Göttingen, Germany). Marker proteins for size exclusion chromatography experiments were purchased from Sigma-Aldrich (Taufkirchen, Germany). Restriction endonucleases and DNA ligase were obtained from ThermoScientific (St. Leon-Rot, Germany) and used as suggested by the manufacturer. Ectoine was a kind gift from the bitop AG (Witten, Germany) and 5-hydroxyectoine was purchased from Merck (Darmstadt, Germany). γ-ADABA was purchased from abcr GmbH (Karlsruhe, Germany).
Kanamycin
Manufactured by Serva Electrophoresis
Sourced in Germany
Kanamycin is an antibiotic used as a selective agent in molecular biology. It inhibits protein synthesis in bacteria, which makes it useful for the selection of cells that have been transformed with plasmids containing the kanamycin resistance gene.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Rifampicin, by Serva Electrophoresis (2 mentions)
Ampicillin, by Serva Electrophoresis (2 mentions)
Chloramphenicol, by Serva Electrophoresis (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Gentamycin, by Serva Electrophoresis (1 mentions)
5 hydroxyectoine, by Merck Group (1 mentions)
Restriction endonuclease, by Thermo Fisher Scientific (1 mentions)
Dna ligase, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Carl Roth (1 mentions)
Glutamax, by Merck Group (1 mentions)
Transferrin, by Merck Group (1 mentions)
Mi plasmid miniprep kit, by Metabion (1 mentions)
2 oxoglutarate, by Merck Group (1 mentions)
X gal, by AppliChem (1 mentions)
Acrylamide solution, by Bio-Rad (1 mentions)
Picoline borane, by Merck Group (1 mentions)
Methanol, by Avantor (1 mentions)
8 protocols using kanamycin
1
Antibiotics and Chemicals Used in Research
2
Naïve B Cell Differentiation Protocol
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Sorted naïve B cells were seeded at 3 × 104/cm2 and cocultured with irradiated cells expressing CD40L at a 10:1 ratio. Cells were cultured in B cell medium: complete RPMI plus 10% FBS, 1% sodium pyruvate, 1% nonessential amino acids, 1% penicillin-streptomycin (Thermo Fisher), 1% GlutaMAX, 0.1% 2-mercaptoethanol, 0.02 ng/mL transferrin (Sigma), and 0.1 mg/mL kanamycin (Serva). Cells were activated with 25 ng/mL IL-4. Every 3 d cells were restimulated by adding 25 ng/mL IL-4. On the seventh day, cells were reseeded at a density of 106 cells/mL, provided with fresh CD40L expressing cells, and activated with 25 ng/mL IL-4 every 3 d. On the 14th day, cells were collected, sorted as described above and immediately processed by either extracting total RNA or genomic DNA.
3
Construction of pMKPccdB Vectors
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To generate pMKPccdB vector with BsaI, BsmBI or AarI cloning/restriction sites, Pol-I/ccdB/Pol-II cassettes containing the three different cloning/restriction sites were transferred from the respective pMPccdB vector versions [39 (link)] using “FastDigest” BsrBI enzyme (Thermo Scientific, USA) at 37°C for 15 min. The gel-purified cassettes were ligated into the EcoRV-blunt ends of pSMART-LC-Kan vector using T4 DNA ligase (Thermo Scientific, USA) according to the manufacture instructions. The ligation reactions were then transformed into “One Shot ccdB Survival 2 T1R” competent E. coli cells (Invitrogen, USA) according to the manufacture instructions and cultured on LB agar plates. 16–18 h later, five individual colonies were picked up and cultured for 18 h at 37°C in 5 ml of LB media containing 30 μg/ml of Kanamycin (SERVA, Germany) followed by plasmid DNA extraction using mi-Plasmid Miniprep Kit (Metabion, Germany). The bacterial culture containing the correct plasmid DNA construct, confirmed by enzymatic digestion and sequencing, was subjected to large scale culture (250 ml LB media) and plasmid DNA extraction/purification using NucleoBond Xtra Maxi (Macherry-Nagel, Germany).
4
Ectoine and 5-Hydroxyectoine Compound Analysis
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Ectoine and 5-hydroxyectoine were kind gifts from Dr. Thomas Lentzen and Dr. Irina Bagyan (bitop AG, Witten, Germany). 2-oxoglutarate (disodium salt) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Anhydrotetracycline-hydrochloride (AHT), desthiobiotin, and Strep-Tactin Superflow chromatography material were purchased from IBA GmbH (Göttingen, Germany). X-Gal was obtained from AppliChem (Darmstadt, Germany), and the antibiotics kanamycin and ampicillin were purchased from Serva Electrophoresis GmbH (Heidelberg, Germany) and Carl Roth GmbH (Karlsruhe, Germany).
5
Preparation of Bacterial Growth Media
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For all bacterial liquid cultures lysogeny broth (LB) was used as a medium. LB was prepared by dissolving 25 g LB Broth (Miller) powder (Sigma Aldrich, USA) in 1 l of MilliQ water, adjusting the pH to 7.0 with NaOH, and autoclaving. LB agar was prepared by adding 1.5% Select agar powder (Sigma Aldrich, USA) to the LB medium and autoclaving. After autoclaving the LB agar was left to cool before adding antibiotics and pouring 20 ml into sterile culture plates (VWR, Germany). Ampicillin (SERVA Electrophoresis, Germany), kanamycin (SERVA Electrophoresis, Germany), or chloramphenicol (SERVA Electrophoresis, Germany) were used at concentrations of 100 µg/ml, 50 µg/ml, and 34 µg/ml respectively when necessary.
6
EF-Tu Expression and Purification Protocol
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EF-Tu (wild type) was prepared from E. coli MRE-600 (ATCC29417) purchased as freeze-dried pellet from UAB School of Medicine, Birmingham, AL, USA. Cells were grown in enriched medium and harvested at mid-logarithmic growth phase. E. coli BL21 (DE3) (Merck Millipore) strain was used for the overexpression of EF-Tu and EF-Tu mutants. For overexpressed EF-Tu, tufA gene from E. coli BL21(DE3) was cloned into the expression vector pet24(+)(kanamycin resistance cassette, C-terminal His-tag; Novagen) using NdeI and XhoI. Cells were grown at 37°C in Terrific broth medium in a Biostat B-plus 5 L fermenter (Sartorius) in the presence of 30 μg ml−1 kanamycin (Serva Electrophoresis). Protein expression was initiated at ∼0.7–0.8 OD600 by addition of 1 mM IPTG (Roth) for 2 h and cells were harvested by centrifugation after 4 h of induction. E. coli W3110 (K12) (Deutsche Sammlung von Mikroorganismen und Zellkulturen) was used to generate the chromosome-encoded C-terminally His-tagged EF-Tu (27 (link)). Cells were grown in LB medium at 37°C up to ∼1 OD600 and harvested by centrifugation. For DDA analysis cells were grown to 0.3 OD600 and treated with streptomycin (4 μM) for 2 h.
7
Protein Purification Using Nickel Affinity Chromatography
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Picoline borane, DMEM cell culture media, and phosphate buffered saline (PBS, pH 7.4) were from Sigma-Aldrich (St Louis, MO). Polyethylene-glycol was purchased from Merck (Kenilworth, NJ). Fetal bovine serum, trypsin-EDTA, and 3-aminopropyltriethoxysilane (APTES) were from Thermo Fisher Scientific (Waltham, MA). Glutaraldehyde was purchased from Carl Roth Chemicals (Karlsruhe, Germany). Methanol and absolute ethanol were purchased from VWR (Radnor, PA). Hydrochloric acid, sulfuric acid, and dry toluene were purchased from Molar Chemicals Kft. (Halasztelek, Hungary). Kanamycin was from SERVA Electrophoresis GmbH (Heidelberg, Germany). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was from Biosynth AG (Staad, Switzerland). The SDS PAGE gel was made from 40% acrylamide solution from Bio-Rad (Hercules, CA) and HPLC grade water in a ratio of 37.5:1. Buffer “A” contained NaH2PO4 from Merck (Kenilworth, NJ) and NaCl from VWR (Radnor, PA). Imidazole for buffer “B” was purchased from Merck.
8
Bacterial Strain Maintenance and Cultivation
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Pseudomonas syringae pv. maculicola strain M2
(RifR) was a kind gift from Dr. Jeffrey L. Dangl (Ritter and Dangl, 1995 (link)), and PsmMut8 was obtained in this
work. E. coli S17-1 λpir (thi pro hsdR hsdMΔrecA RP4-2traTc::Mu Km::Tn7)
(de Lorenzo et al., 1990 (link))
was obtained from Dr. Kate J. Wilson. E. coli DH5α competent cells
(supE44ΔlacU169 (f80 lacZ DM15)
hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (Sambrook and Russell, 2001 ) were used for cloning
experiments. A SwaI restriction site was added into the
SmaI site on pUIRM504 (Marsch-Moreno et al., 1998 (link)) to form the plasmid pMDC505
(unpublished results); with this change, the transposable element
pTn5cat (Marsch-Moreno et
al., 1998 ) was modified into
pTn5cat1. King’s B medium (King et
al., 1954), minimal medium M9 (Sambrook
and Russell, 2001 ) or M9CA (Difco) was used to culture P. s.
maculicola strains and in the assay to determine the conditions for
cat expression, with or without the additions described below. LB
medium was used to culture the E. colistrains. Chloramphenicol,
rifampicin and kanamycin were purchased from Serva or Sigma-Aldrich Chemicals.
+ Expand
Pseudomonas syringae pv. maculicola strain M2
(RifR) was a kind gift from Dr. Jeffrey L. Dangl (Ritter and Dangl, 1995 (link)), and PsmMut8 was obtained in this
work. E. coli S17-1 λpir (thi pro hsdR hsdMΔrecA RP4-2traTc::Mu Km::Tn7)
(de Lorenzo et al., 1990 (link))
was obtained from Dr. Kate J. Wilson. E. coli DH5α competent cells
(supE44ΔlacU169 (f80 lacZ DM15)
hsdR17 recA1 endA1 gyrA96 thi-1 relA1) (Sambrook and Russell, 2001 ) were used for cloning
experiments. A SwaI restriction site was added into the
SmaI site on pUIRM504 (Marsch-Moreno et al., 1998 (link)) to form the plasmid pMDC505
(unpublished results); with this change, the transposable element
pTn5cat (
al., 1998
pTn5cat1. King’s B medium (King et
al., 1954), minimal medium M9 (
and Russell, 2001
maculicola strains and in the assay to determine the conditions for
cat expression, with or without the additions described below. LB
medium was used to culture the E. colistrains. Chloramphenicol,
rifampicin and kanamycin were purchased from Serva or Sigma-Aldrich Chemicals.
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