arcticexpress de3 competent e coli, by Agilent Technologies - Product details

1

Cloning and Expression of ctxIII Protein

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Genomic DNA was prepared using a genomic DNA purification kit (Promega, Madison, WI). ctxIII DNA was amplified and pieced together through PCR, subcloned onto pBluescript, and the sequence confirmed. The DNAs of N-terminal FLAG-tagged (DYKDDDDKENLYFQG-) full-length ctxIII, N-terminal-ctxIII (residues 1–309) and C-terminal-ctxIII (residues 310–489) were cloned to the pTIKL expression vector. N-terminal GFP-tagged full-length ctxIII was cloned to the DM317 vector for expression of the proteins in Dictyostelium. FLAG-ctxIII was also cloned into pFaxtBac1 for expression in SF-9 cells. N-terminal His-tagged (MRGSHHHHHHIPIEGR-) ctxI and ctxII (generous gifts of Jan Faix, Hannover Medical School, Hannover, Germany) and FLAG-ctxIII were cloned into pETDuet-1 (Novagen, Madison, WI) for bicistonic expression in Arctic Express (DE3)–competent E. coli (Agilent, Palo Alto, CA).

Liu X., Shu S., Yu S., Lee D.Y., Piszczek G., Gucek M., Wang G, & Korn E.D. (2014). Biochemical and biological properties of cortexillin III, a component of Dictyostelium DGAP1–cortexillin complexes. Molecular Biology of the Cell, 25(13), 2026-2038.

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Monocolonization of Germ-free Mice with E. coli

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E. coli containing plasmid pOPS0750, a gift from Philip Poole (Addgene plasmid # 115511; http://n2t.net/addgene:115511; RRID:Addgene_115511) was grown in Luria Bertani (LB) broth + Kanamycin at 20 μg/mL overnight. Plasmid DNA was extracted using a Qiaprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions and DNA concentration measured using a Qubit dsDNA kit (ThermoFisher). ArcticExpress (DE3) Competent E. coli (Agilent) were transformed (50 ng of plasmid) per the instructions provided. Transformed bacteria were plated on LB agar plates with Kanamycin (20 μg/mL) and Tetracycline (20 μg/mL) and incubated at 37°C overnight. Individual colonies were streaked for isolation with single colonies picked and then grown in LB broth. Overnight cultures were spun for 5 min at max speed and resuspended to an OD₆₀₀ of 1.0 (10^9 cells/mL) in sterile PBS. 200 μL of transformed bacteria, or DE3 competent E. coli were used to monocolonize 10-week old female germ-free Swiss Webster Mice in a random order. Confirmation of monocolonization by either control or GUS⁺E. coli was done by collecting pellets at day 7, and inoculating pellets in selective media at 37°C, examining for growth overnight. Pellets from culture positive, monocolonized animals were then used to assess fecal PA as described earlier in the methods.

Edwinson A.L., Yang L., Peters S., Hanning N., Jeraldo P., Jagtap P., Simpson J.B., Yang T.Y., Kumar P., Mehta S., Nair A., Breen-Lyles M., Chikkamenahalli L., Graham R.P., De Winter B., Patel R., Dasari S., Kashyap P., Griffin T., Chen J., Farrugia G., Redinbo M.R, & Grover M. (2022). Gut Microbial β-Glucuronidases Regulate Host Luminal Proteases and are depleted in Irritable Bowel Syndrome. Nature microbiology, 7(5), 680-694.

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Monocolonization of Germ-free Mice with E. coli

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E. coli containing plasmid pOPS0750, a gift from Philip Poole (Addgene plasmid # 115511; http://n2t.net/addgene:115511; RRID:Addgene_115511) was grown in Luria Bertani (LB) broth + Kanamycin at 20 μg/mL overnight. Plasmid DNA was extracted using a Qiaprep Spin Miniprep Kit (Qiagen) according to manufacturer’s instructions and DNA concentration measured using a Qubit dsDNA kit (ThermoFisher). ArcticExpress (DE3) Competent E. coli (Agilent) were transformed (50 ng of plasmid) per the instructions provided. Transformed bacteria were plated on LB agar plates with Kanamycin (20 μg/mL) and Tetracycline (20 μg/mL) and incubated at 37°C overnight. Individual colonies were streaked for isolation with single colonies picked and then grown in LB broth. Overnight cultures were spun for 5 min at max speed and resuspended to an OD₆₀₀ of 1.0 (10^9 cells/mL) in sterile PBS. 200 μL of transformed bacteria, or DE3 competent E. coli were used to monocolonize 10-week old female germ-free Swiss Webster Mice in a random order. Confirmation of monocolonization by either control or GUS⁺E. coli was done by collecting pellets at day 7, and inoculating pellets in selective media at 37°C, examining for growth overnight. Pellets from culture positive, monocolonized animals were then used to assess fecal PA as described earlier in the methods.

Edwinson A.L., Yang L., Peters S., Hanning N., Jeraldo P., Jagtap P., Simpson J.B., Yang T.Y., Kumar P., Mehta S., Nair A., Breen-Lyles M., Chikkamenahalli L., Graham R.P., De Winter B., Patel R., Dasari S., Kashyap P., Griffin T., Chen J., Farrugia G., Redinbo M.R, & Grover M. (2022). Gut Microbial β-Glucuronidases Regulate Host Luminal Proteases and are depleted in Irritable Bowel Syndrome. Nature microbiology, 7(5), 680-694.

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Arcticexpress de3 competent e coli

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3 protocols using arcticexpress de3 competent e coli

Cloning and Expression of ctxIII Protein

Monocolonization of Germ-free Mice with E. coli

Monocolonization of Germ-free Mice with E. coli

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