Bl21 de3 ripl

Manufactured by Agilent Technologies

The BL21(DE3)-RIPL is a competent E. coli strain developed by Agilent Technologies for protein expression. It is designed to optimize the expression of recombinant proteins in bacterial systems.

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Lab products found in correlation

Ubiquitin, by R&D Systems (1 mentions) Cobalt beads, by Thermo Fisher Scientific (1 mentions) Mg atp, by R&D Systems (1 mentions) Flag peptide, by Merck Group (1 mentions) M2 beads, by Merck Group (1 mentions) Glutathione sepharose 4b resin, by GE Healthcare (1 mentions) Complete without edta, by Roche (1 mentions) Dnase 1, by Thermo Fisher Scientific (1 mentions) Rnase a, by Roche (1 mentions) Arcticexpress de3, by Agilent Technologies (1 mentions)

5 protocols using bl21 de3 ripl

Linear Ubiquitination Assay Protocol

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ASC-Flag and NLRP3-Flag were purified from 293T cells by M2 beads (Sigma-Aldrich) in high salt buffer (500 mM NaCl, 1% NP-40, and 50 mM Tris, pH 8.0) and eluted with Flag peptide (Sigma-Aldrich), followed by 10 kD MWCO dialysis in excess TBS overnight. HOIL-1L-6xHIS and HOIP-6xHIS were cloned into the pET-duet-1 vector, coexpressed in BL21 DE3 RIPL (Agilent Technologies) bacteria, and purified from cultures grown in the presence of 0.2 mM ZnCl₂ and 0.5 mM IPTG at 16°C for 24 h. Cells were lysed in native buffer (50 mM Tris, PH 8.0, and 100 mM NaCl) and HOIL-1L/HOIP were co-purified on cobalt beads (Thermo Fisher Scientific) with 150 mM imidazole elution and overnight dialysis in lysis buffer. HOIL-1L/HOIP (LUBAC) purification preps were stored at 4°C and never frozen. E1 (Ube1), E2 (UbcH5c), and ubiquitin were purchased from Boston Biochem. In vitro linear ubiquitination assays contained 200 ng E1, 400 ng E2, 1 µg HOIL-1L/HOIP-6xHIS purification prep, 20 mM Tris, pH 7.5, 5 mM DTT, 5 mM MgCl₂, 2 mM MgATP (Boston Biochem), 10 µg ubiquitin, and 0.5 µg purified Flag-tagged substrate.

Rodgers M.A., Bowman J.W., Fujita H., Orazio N., Shi M., Liang Q., Amatya R., Kelly T.J., Iwai K., Ting J, & Jung J.U. (2014). The linear ubiquitin assembly complex (LUBAC) is essential for NLRP3 inflammasome activation. The Journal of Experimental Medicine, 211(7), 1333-1347.

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Purification of PPH-4.1 Phosphatase Enzyme

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The cDNA of pph-4.1 was cloned into the GST-fusion expression vector pGEX-4T1 and the resulting plasmid transformed into BL21(DE3)-RIPL (Agilent). The resulting bacterial strain was grown to an OD₆₀₀ of 0.6 at 37 °C and 230 rpm and then induced with IPTG for 18 h at 18 °C and 230 rpm. Next the bacteria were harvested and frozen in liquid nitrogen. For the purification of PPH-4.1, the bacteria were thawed into TBS buffer containing 1 mM EDTA, 1 mM DTT, and Complete without EDTA (Roche), and incubated with lysozyme for 30 min at 4 °C. Resulting lysate was sonicated, Triton X-100, RNase A (Roche), and DNase I (Invitrogen) were added to concentrations of 1% (v/v), 10 µg ml⁻¹, and 5 µg ml⁻¹, respectively, and the lysate then incubated for another 30 min at 4 °C. Glutathione-Sepharose 4B resin (GE Healthcare) was added to the lysate and incubated rotating for 2 h at 4 °C. Finally, the resin was washed in TBS, resuspended in TBS, and then used for in vitro phosphatase assays.

Sen I., Zhou X., Chernobrovkin A., Puerta-Cavanzo N., Kanno T., Salignon J., Stoehr A., Lin X.X., Baskaner B., Brandenburg S., Björkegren C., Zubarev R.A, & Riedel C.G. (2020). DAF-16/FOXO requires Protein Phosphatase 4 to initiate transcription of stress resistance and longevity promoting genes. Nature Communications, 11, 138.

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Yeast and Bacterial Strain Protocols

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Saccharomyces cerevisiae strains (WT, Δcoq9 and Δcoq7) were the haploid MATalpha BY4742 (his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) from the gene deletion consortium (Thermo #YSC1054). Escherichia coli strains for protein overexpression were BL21[DE3]-RIPL and ArcticExpress (DE3) from Agilent (Cat#230280 and Cat#230192 respectively). Escherichia coli strain for DNA cloning and mutagenesis was DH5α from NEB (Cat#C2987). Please see individual methods for detailed culture conditions.

Manicki M., Aydin H., Abriata L.A., Overmyer K.A., Guerra R.M., Coon J.J., Peraro M.D., Frost A, & Pagliarini D.J. (2022). Structure and functionality of a multimeric human COQ7:COQ9 complex. Molecular cell, 82(22), 4307-4323.e10.

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Yeast and E. coli Strains Protocol

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Saccharomyces cerevisiae strains (WT and ∆coq9) were the haploid MATalpha BY4742
(his3∆1 leu2∆0 lys2∆0 ura3∆0) from the gene deletion consortium (Thermo #YSC1054). Escherichia
coli strains for protein overexpression were BL21[DE3]-RIPL from Agilent and for cloning applications, DH5α
from NEB were used. Unless specified otherwise, E. coli were grown at 37 °C in LB media with antibiotics
and S. cerevisiae were grown at 30 °C in drop-out minimal media.

Lohman D.C., Aydin D., Von Bank H.C., Smith R.W., Linke V., Weisenhorn E., McDevitt M.T., Hutchins P., Wilkerson E.M., Wancewicz B., Russell J., Stefely M.S., Beebe E.T., Jochem A., Coon J.J., Bingman C.A., Peraro M.D, & Pagliarini D.J. (2019). An isoprene lipid binding protein promotes eukaryotic coenzyme Q biosynthesis. Molecular cell, 73(4), 763-774.e10.

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Cloning and Expression of daf-16b and hlh-30a

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An HA₄ tag followed by the cDNA of daf-16b was cloned into the GST-fusion expression vector pGEX-4T1, and a myc₆ tag followed by the cDNA of hlh-30a was cloned into the His₆-fusion expression vector pET-28a(+). Empty pGEX-4T1, expressing GST alone, was used as a negative control. All three plasmids were transformed into BL21(DE3)-RIPL (Agilent). Cultures of the resulting bacterial strains were grown to an OD of 0.6-0.7 at 37 °C and then induced with IPTG for 18–20 h at 18 °C. Eventually, the bacteria were harvested and frozen in liquid nitrogen.

Lin X.X., Sen I., Janssens G.E., Zhou X., Fonslow B.R., Edgar D., Stroustrup N., Swoboda P., Yates JR 3.r.d., Ruvkun G, & Riedel C.G. (2018). DAF-16/FOXO and HLH-30/TFEB function as combinatorial transcription factors to promote stress resistance and longevity. Nature Communications, 9, 4400.

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