Plasmids were transformed into E. coli strain BL21(DE3) (TRANSGEN Biotech., Beijing, China). Single colonies were inoculated 2XYT medium with antibiotics at 37 °C. Overnight culture was transferred to fresh medium at 1:100 ratio. When OD600 reached between 0.4 to 0.6, IPTG (1 mm final) was added to induce expression of GFP1-9. Mandipropamid or DMSO (solvent control) was also added. The culture was incubated for 3 h, and 100 µL of each sample was transferred to a 96 well plate. Both GFP florescence (Ex: 488, Em: 510) and OD600 was measured by the Perkin Elmer Enspire™ 2,300 Multilabel Reader. After the values for medium were subtracted, florescence over OD600 was calculated and compared to that of the DMSO control sample.
E coli bl21 de3 strain
Manufactured by Transgene
Sourced in China
E. coli BL21 (DE3) strain is a commonly used bacterial host strain for the expression of recombinant proteins. It is a derivative of the E. coli B strain that contains the DE3 lysogen, which carries the T7 RNA polymerase gene under the control of the lacUV5 promoter. This strain is designed for high-level protein expression, particularly for proteins that may be toxic or difficult to express in other E. coli strains.
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5 protocols using e coli bl21 de3 strain
1
GFP Expression Induction Assay
2
Codon-optimized HE-ORF2 and HA-VP1 Antigens
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The synthetic HE-ORF2-tuftsin and HA-VP1-tuftsin antigen genes, encoding aa 368–607 of HE-ORF2 linked to tuftsin and aa 1–198 of HA-VP1 linked to tuftsin, respectively, were codon-optimized for expression in Escherichia coli. By PCR amplification using Pfu DNA polymerase (Promega, Madison, WI, USA), two genetic constructs were prepared for the expression of HE-ORF2 (aa 368–607) or HA-VP1 (aa 1–198) in E. coli without tuftsin as a control plasmid. The specific primers for HE-ORF2 synthesized by Sangon Biotech (Shanghai, China) were 5’-GGAATTCCATATGATCGCTCT-3’ (forward) and 5’-GGAATTCCATATGATCGCTCT-3’ (reverse). The specific primers for HA-VP1 were 5’-GGAATTCCATATGGTTGGTGACG-3’ (forward) and 5’-GGAATTCCATATGATCGCTCT-3’ (reverse). After an initial denaturation at 94°C for 5 min, all reactions were subjected to 35 cycles at 94°C for 30 s, 56°C for 30 s and 72°C for 45 s, followed by a final extension at 72°C for 5 min. After double-enzyme digestion with Xho I and Nde I, the products were cloned into the pET43a vector (Novagen, Billerica, MA, USA) and transformed into E. coli strain BL21 (DE3) (TransGen Biotech, Beijing, China). Ampicillin-resistant colonies were selected and identified by restriction endonuclease analysis of the plasmids as well as sequencing.
3
Cloning and Purification of Recombinant BoHSP90 Proteins
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The ORFs of BoHSP90-A and BoHSP90-B were amplified from the cDNA and subcloned into the pET-32a vector using the EcoRI and XhoI restriction sites. The resulting plasmids pET-32a/BoHSP90-A and pET-32a/BoHSP90-B were transformed into the E. coli BL21™ (DE3) strain (Transgene, China) using the standard method. The plasmids from bacterial cells at each step were extracted using QIAGEN Plasmid Mini Kit (Qiagen, Hilden, Germany) and subjected to restriction enzyme digestion and sequencing analysis in order to verify successful cloning and the orientation of genes in the vector. The proteins were expressed in E. coli by inducing with 1 mM isopropyl β-D-thiogalactoside (IPTG). The rBoHSP90-A and rBoHSP90-B were expressed as His-fusion proteins and their expressions were monitored through SDS-PAGE. The uninduced plasmids pET-32a/BoHSP90-A and pET-32a/BoHSP90-B and IPTG induced empty vector (pET-32a) were used as negative controls (Figure 1 ).
The rBoHSP90-A and rBoHSP90-B were expressed in the insoluble-fraction of E. coli and were purified using HiTrap TALON crude (GE Healthcare, Sweden). The affinity chromatography was performed according to the manufacturer’s instructions. The quantities of recombinant proteins were measured by BCA protein assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.
The rBoHSP90-A and rBoHSP90-B were expressed in the insoluble-fraction of E. coli and were purified using HiTrap TALON crude (GE Healthcare, Sweden). The affinity chromatography was performed according to the manufacturer’s instructions. The quantities of recombinant proteins were measured by BCA protein assay kit (Beyotime Institute of Biotechnology, China) according to the manufacturer’s instructions.
4
E. coli Protein Expression and Purification
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E. coli BL21 (DE3) strain (TransGen Biotech, Beijing, China) was used for protein expression [58 (link)]. The plasmids of G6046 or its truncations were transformed into E. coli BL21 (DE3) strain, and the single clone was amplified to 1 L LB medium (100 μg/mL kanamycin or 100 μg/mL ampicillin). The cells were cultured at 37 °C and IPTG was added to a final concentration of 0.5 mM when OD600 = 0.8. Cells were incubated for another 20 h at 16 °C. The cells were harvested by centrifugation and stored at −40 °C for further use.
The recombinant G6046 protein was purified using the affinity chromatography method. The cells were re-suspended in the lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol) and then disrupted by sonication for 30 min (5 s on, 10 s off) on ice. The cell lysate was centrifuged at 16,000× g for 1 h. Two milliliters of Ni-NTA resin was added to a gravity column and equilibrated with the lysis buffer. The supernatant was loaded onto the column and washed with 50 mL of the lysis buffer containing 20 mM imidazole. The protein was eluted with 20 mL of the lysis buffer supplemented with 200 mM imidazole. The concentration of eluted G6046 protein was measured using the Bradford assay (TIANGEN biotech Beijing Co., Ltd., Beijing, China) and the purity was evaluated by SDS-PAGE.
The recombinant G6046 protein was purified using the affinity chromatography method. The cells were re-suspended in the lysis buffer (50 mM Tris, pH 7.5, 100 mM NaCl, 10% glycerol) and then disrupted by sonication for 30 min (5 s on, 10 s off) on ice. The cell lysate was centrifuged at 16,000× g for 1 h. Two milliliters of Ni-NTA resin was added to a gravity column and equilibrated with the lysis buffer. The supernatant was loaded onto the column and washed with 50 mL of the lysis buffer containing 20 mM imidazole. The protein was eluted with 20 mL of the lysis buffer supplemented with 200 mM imidazole. The concentration of eluted G6046 protein was measured using the Bradford assay (TIANGEN biotech Beijing Co., Ltd., Beijing, China) and the purity was evaluated by SDS-PAGE.
5
Heterologous Expression of Plant CDK Proteins
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Plasmids pCold-TF-TaICK1, pCold-TF-TaCYCD2;1, and pCold-TF-TaCYCD6;1 were introduced into the E. coli BL21 (DE3) strain (TransGen Biotech, China) via the heat shock method, as described by the manufacturer. Colonies of verified transformants were incubated in LB [73 (link)] medium containing 50 mg L−1 carbenicillin at 37 °C for 8–12 h until the optical density OD600 reached 0.6–0.8. IPTG (Isopropyl β-D-Thiogalactoside) was then added into the medium at a final concentration of 0.5 mM, followed by incubation at 16 °C overnight with shaking at 100 rpm. The control culture was treated using the same protocol without IPTG. After incubation, the crude cell extracts were prepared as described [74 (link)]. Then, the crude proteins were analyzed using SDS-PAGE and dyed with Coomassie brilliant blue R-250 using the protocol of Sambrook and Russell [74 (link)].
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