Deuterium oxide (99.96%) was from Euriso-Top (Saint-Aubin Cedex, France). U , U MP, uracil, K2HPO4, KH2PO4, NaHCO3, MnCl2, HEPES, NaCl, IPTG, imidazole, tetra-n-butylammonium bromide, glycerol, and ATP were from Carl Roth (Karlsruhe, Germany). Glc1,6diP and SYPRO orange, was from Sigma Aldrich (St. Louis, USA). Rib5P was purchased from Biosynth (Staad, Switzerland). Expression vectors (pet15b or pet28a+) containing the genes for YeiN, DeoB, UP, and Yjjg were from Genescript (Leiden, The Netherlands).
Isopropyl β d 1 thiogalactopyranoside (iptg)
Manufactured by Carl Roth
Sourced in Germany
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a laboratory chemical commonly used as an inducer in bacterial expression systems. It acts by binding to and inactivating the lac repressor protein, allowing for the expression of genes under the control of the lac promoter.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Carl Roth (2 mentions)
Pqe30, by Qiagen (2 mentions)
Hitrap, by Cytiva (2 mentions)
L arabinose, by Carl Roth (2 mentions)
Ampicillin, by Merck Group (2 mentions)
Sypro orange, by Merck Group (1 mentions)
Sodium chloride (nacl), by Carl Roth (1 mentions)
Deuterium oxide, by Eurisotop (1 mentions)
K2hpo4, by Carl Roth (1 mentions)
Kh2po4, by Carl Roth (1 mentions)
Nahco3, by Carl Roth (1 mentions)
Mncl2, by Carl Roth (1 mentions)
Adenosine triphosphate (atp), by Carl Roth (1 mentions)
Uracil, by Carl Roth (1 mentions)
Glycerol, by Carl Roth (1 mentions)
Hepes, by Carl Roth (1 mentions)
Ni nta agarose, by Thermo Fisher Scientific (1 mentions)
Cnbr activated sepharose 4b, by GE Healthcare (1 mentions)
Ktaprime plus, by GE Healthcare (1 mentions)
Prep4, by Qiagen (1 mentions)
25 protocols using isopropyl β d 1 thiogalactopyranoside (iptg)
1
Purification and Characterization of Bacterial Enzymes
2
Purification of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All reagents used were of the highest purity available. NADPH was from Biomol, Hamburg. Imidazole, IPTG, and 1,4-dithiothreitol (DTT) were obtained from Roth, Karlsruhe, and Ni-NTA agarose from Invitrogen, Karlsruhe. PCR primers were from MWG-Biotech, Ebersberg; the vector pQE30 and the E. coli host strain M15 were from Qiagen, Hilden. CNBr-activated Sepharose 4B (Amersham) was purchased from GE Healthcare, Munich.
3
Purification of Recombinant Anthrax Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
Escherichia coli BL21-CodonPlus-RIL cells (Stratagene, La Jolla, CA) harboring the plasmid pREP 4 and pQE-30 (both Qiagen, Venlo, Netherland) encoding either rPA83 or rBclA coupled to a HIS-tag were grown and purified as described previously [34 (link)]. Briefly, bacterial inoccula were grown in antibiotic supplemented Standard medium I (Roth, Karlsruhe, Germany). Upon reaching OD600nm of 0.6, protein production was induced with IPTG (Roth, Karlsruhe, Germany). After incubation for 3 h at 37 °C shaking cultures were lysed and proteins purified on a HiTrap Sepharose column loaded with nickel(II)sulfate (Sigma-Aldrich, Taufkirchen, Germany) via FPLC with Äktaprime plus (GE Healthcare, Freiburg, Germany). After dialysis onto 5 mM HEPES-buffer (Roth, Karlsruhe, Germany) and quality assessment via SDS-page, protein yield was established via Bicinchoninsäure test (Interchim, Montlucon, France). Proteins used for ELISA received no further treatment while proteins used for vaccination were tested for endotoxin (ET) yield using the Limulus Amoebocyte Lysate Endochrome-K test kit (Charles River, Wilmington, MA) according to the manufactures’s instructions. Endotoxin removal was performed via EndoTrap blue endotoxin removal system (Hyglos, Bernried, Germany) for proteins with ET values more than 50 ng/kg body weight.
+ Expand
Escherichia coli BL21-CodonPlus-RIL cells (Stratagene, La Jolla, CA) harboring the plasmid pREP 4 and pQE-30 (both Qiagen, Venlo, Netherland) encoding either rPA83 or rBclA coupled to a HIS-tag were grown and purified as described previously [34 (link)]. Briefly, bacterial inoccula were grown in antibiotic supplemented Standard medium I (Roth, Karlsruhe, Germany). Upon reaching OD600nm of 0.6, protein production was induced with IPTG (Roth, Karlsruhe, Germany). After incubation for 3 h at 37 °C shaking cultures were lysed and proteins purified on a HiTrap Sepharose column loaded with nickel(II)sulfate (Sigma-Aldrich, Taufkirchen, Germany) via FPLC with Äktaprime plus (GE Healthcare, Freiburg, Germany). After dialysis onto 5 mM HEPES-buffer (Roth, Karlsruhe, Germany) and quality assessment via SDS-page, protein yield was established via Bicinchoninsäure test (Interchim, Montlucon, France). Proteins used for ELISA received no further treatment while proteins used for vaccination were tested for endotoxin (ET) yield using the Limulus Amoebocyte Lysate Endochrome-K test kit (Charles River, Wilmington, MA) according to the manufactures’s instructions. Endotoxin removal was performed via EndoTrap blue endotoxin removal system (Hyglos, Bernried, Germany) for proteins with ET values more than 50 ng/kg body weight.
4
Blue Light Responsive RFP Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In a 48‐well plate (Corning), 0.5 mL cultures were prepared, using LB media, supplemented with 50 µg mL−1 chloramphenicol and 25 µg mL−1 of kanamycin, 400 µm IPTG and 25 mm L‐arabinose (all Carl Roth). The wells were inoculated with 5 µL of precultures that had been prepared as described above. The samples were then incubated at 37 °C and 220 rpm for 3 h in darkness, followed by 3 h incubation under blue light exposure and a final step of 3 h in the dark. Prior to the first incubation step and after each following incubation period, the RFP fluorescence and the OD600 were measured in a plate reader. Following every incubation period the samples were diluted 1:30 into new plates with pre‐warmed fresh media, containing all supplements. The final relative fluorescence was obtained by normalizing the RFP values to the measured OD600. Three independent replicates were generated by repeating experiments on different days.
5
Recombinant Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemically competent E. coli M15 [pREP4] cells (Qiagen) were transformed with the pQE-9 expression vector containing the gene construct of interest (Cys-SpyCatcher or eGFP-SpyTag). Transformed cells were plated on LB agar plates with 200 µg/ml ampicillin and 25 µg/ml kanamycin. Single colonies were picked for inoculating an overnight culture in liquid LB with antibiotics (10 g NaCl, 10 g Bactotrypton, 5 g yeast extract per 1 l; 200 µg/ml ampicillin and 25 µg/ml kanamycin) and incubated over night at 28 °C. Appropriate amounts of the overnight culture were used to inoculate a 1 l expression culture to an optical density (OD600) of 0.02 and the best time point of induction was determined (see Additional file 7 : Fig. S4). The expression cultures were cultivated at 28 °C and shaking at 180–200 rpm until an OD600 of 0.5 was reached. Recombinant protein expression was induced by addition of 0.5 mM IPTG (Roth) for 4 h at 28 °C and shaking at 180–200 rpm. After 4 h, cells were harvested by centrifugation at 5000g for 20 min at 4 °C. Cell pellets were frozen at – 20 °C for storage.
6
Periplasmic Protein Expression in E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression was performed in E. coli BL 21 codonplus using a pET-22b vector system with a pelB-leader sequence for export to the periplasm of E.coli. After transformation cells were cultivated at 180 rpm and 30°C in shaking flasks in 400 mL LB-medium. After the OD600 had reached 0.4, expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG, Carl Roth, final concentration 0.2 mM). The expression proceeded over 15 h at 20°C. The cells were harvested by centrifugation at 4700 rpm for 20 min at 4°C (Heraeus Multifuge X3 FR). Protein purification from the periplasm was carried out by osmotic shock in ddH2O, using the protocol by Petersen et al. [53 (link)]. The supernatant was frozen in liquid nitrogen and lyophilized overnight (Lyophilizer, Beta 1–8 Martin Christ). The product was analyzed by SDS-polyacrylamide-gel electrophoresis (SDS-PAGE, S2 Fig ).
7
Protein Expression in E. coli BL21(DE3) Rosetta
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E. coli BL21(DE3) Rosetta cells (SGC Frankfurt) were transformed with the respective E. coli expression plasmids for protein production. Cells were grown at 37 °C in 2xYT medium supplemented with 100 µM zinc acetate (Carl Roth) to an optical density (OD) of ~0.8. Protein expression was induced with 1 mM IPTG (Carl Roth) for 16–18 h at 18 °C. For labelled expression, cells were grown in LB medium at 37 °C to an OD of ~1, harvested by centrifugation and resuspended in M9 minimal medium supplemented with 100 µM zinc acetate and the appropriate isotopic labelling reagents (1 g/l 15NH4Cl (Cambridge Isotope Laboratories Inc.) and 4 g/l glucose (Carl Roth) for 15N-labelling; 1 g/l 15NH4Cl and 2 g/l 13C-glucose (Cambridge Isotope Laboratories Inc.) for 15N/13C-labelling) to an OD of ~0.3. Cells were then grown at 37 °C to an OD of ~0.8 and protein expression was carried out as described above.
8
Purification of pDEST15-UBL-hHR23B Fusion Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pDEST15–UBL–hHR23B was transformed into B834 E. coli (Novagen) according to manufacturer’s instructions. The bacteria were grown at 37°C to an OD600nm of 0.6 and induced for 3 h with 1 mM IPTG (Roth). Pellets of 1 liter bacteria culture were then lysed in 25 ml GSH-binding buffer (GBB) (1x PBS, 10 mM MgCl2, 1 mM DTT) by sonication. Afterwards, the bacteria were centrifuged at 100,000g for 1 h. 500 μl glutathione-Sepharose (GE Healthcare) was added to the supernatant for batch purification. After washing with 75 ml GBB, the beads were stored for further use.
9
Expression and Purification of His-Tagged UIM2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pET26b–His10–UIM2 was transformed into B834 E. coli (Novagen) according to the manufacturer’s instructions. The bacteria were grown at 37°C to an OD600 of 0.6 and induced for 3 h with 1 mM IPTG (Roth). Pellets corresponding to 2 liters bacteria culture were lysed in 25 ml lysis buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0, using NaOH) by sonication. Thereafter, the lysed bacteria were centrifuged at 100,000g for 1 h. Afterwards, 0.5 ml of Ni–IDA resin (Protino) were added to the supernatant for batch purification. After washing with 75 ml of lysis buffer, the protein was eluted by adding 2 ml elution buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0, 300 mM imidazole) for 2 h hours at 4°C while rotating. The imidazole concentration was then reduced to 0.1 mM by ultrafiltration (Ultracel-3K; Merck). The protein was then concentrated to ∼2 mg/ml and stored at −20°C for further use.
10
E. coli Strain MG1655 Growth Conditions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E. coli strain MG1655 (F− λ− ilvG rfb-50 rph-1) [58 (link)] carrying pETgfp-mut2-AGGAGG(3) plasmid [59 (link)] was used. Bacteria were grown in LB-Miller media and in MOPS (morpholinepropanesulfonic acid) media supplemented with 0.1% glucose (MOPS-Glc), as described [60 (link)]. All growth media were supplemented with kanamycin to a final concentration of 25 µg/mL. For overexpression experiments, MG1655 bacteria with and without pBbE6k plasmids [61 (link)] were used. Media was supplemented with IPTG (Isopropyl β-d-1-thiogalactopyranoside) (Roth. Germany) to a final concentration of 1 mM.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!