The 3′-UTR RNA probes of MdOPT3, MxOPT3, and AtOPT3 were transcribed using the In vitro Transcription T7 Kit (Takara, Shiga, Japan; 6140). The GST-tagged RBPs were expressed using the pGEX4T-1 prokaryotic expression vector (cwbiotech, Beijing, China; CW2198) in E. coli BL21 (Transgen, Strasbourg, France; CD901-02). SPR measurements were performed using the GST Capture Kit on the Biacore X100 platform (GE Healthcare Chicago, IL, USA). RNA and RBP binding levels were tested using a Biacore Binding Analysis program (GE Healthcare) with purified RNA probes as the mobile phase, and GST-tagged RBPs as the stationary phase. Binding level is defined as the value of the binding number of the Reaction Unit given by the binding analysis program after the stable combination of the sample and the fixed phase.
E coli bl21
Manufactured by Transgene
Sourced in China
E. coli BL21 is a strain of Escherichia coli bacteria commonly used in molecular biology and biochemistry laboratories. It is a robust host for the expression and production of recombinant proteins. The BL21 strain is designed to facilitate efficient protein expression and simplify the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Escherichia coli dh5α, by Transgene (3 mentions)
In vitro transcription t7 kit, by Takara Bio (1 mentions)
Gst capture kit, by GE Healthcare (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Bradford kit, by Beyotime (1 mentions)
Protein g agarose column, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose column, by Thermo Fisher Scientific (1 mentions)
E coli bl21 de3 competent cell, by Transgene (1 mentions)
Anti his and anti gst antibodies, by Abmart (1 mentions)
His tagged protein purification kit, by CWBIO (1 mentions)
Axyprep dna gel extraction kit, by Corning (1 mentions)
E coli top10, by Transgene (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Pet 30a, by Sangon (1 mentions)
Ni charged resin, by Bio-Rad (1 mentions)
12 protocols using e coli bl21
1
Characterization of OPT3 RNA-Binding Proteins
2
Cultivation and Preservation of Gordonia neofelifaecis
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Gordonia neofelifaecis (NRRL B-59395) was preserved in our laboratory (Ge et al. 2011 (link)) and cultivated in liquid LB medium (Luria–Bertani broth) at 37 °C. The strain had been originally isolated from the faeces of Neofelis nebulosa. Escherichia coli DH5α and E. coli BL21 (purchased from Transgen Biotech Co., Ltd, Beijing, China) were grown in LB broth or Super Optimal Broth (SOB, 2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4) (Hanahan 1983 (link)) at 37 °C/200 rpm. Kanamycin (25 μg/mL) was added to the growth medium when necessary. For growing on solid medium, 2% (w/v) agar was added.
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Gordonia neofelifaecis (NRRL B-59395) was preserved in our laboratory (Ge et al. 2011 (link)) and cultivated in liquid LB medium (Luria–Bertani broth) at 37 °C. The strain had been originally isolated from the faeces of Neofelis nebulosa. Escherichia coli DH5α and E. coli BL21 (purchased from Transgen Biotech Co., Ltd, Beijing, China) were grown in LB broth or Super Optimal Broth (SOB, 2% peptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4) (Hanahan 1983 (link)) at 37 °C/200 rpm. Kanamycin (25 μg/mL) was added to the growth medium when necessary. For growing on solid medium, 2% (w/v) agar was added.
3
Bacterial Strains for Protein Expression
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Escherichia coli DH5α and E. coli BL21 used for protein expression and purification were purchased from TransGen (Beijing, China) and cultured in LB medium. Wild-type L. monocytogenes strain EGD and its hly-deficient mutant EGDΔhly and the complementation strain EGDΔhly::hly were kind gifts from Pascale Cossart (Institut Pasteur, Paris, France) and were grown in Trypticase soy broth (TSB) medium with shaking at 37°C.
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4
Recombinant PoMPO Protein Production
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The sequence (427-1155 bp) with strong antigenicity of PoMPO was amplified using primers PoMPO-F/PoMPO-R (Table 1
The concentration of rPoMPO was determined to be 1 mg mL-1 by using the Bradford kit (P0006C, Beyotime, Shanghai, China). BALB/c mice were immunized with 100 µg rPoMPO according to previously reported methods (29 (link)). After two boosters with a mixture of rPoMPO and incomplete Freund’s adjuvant (Sigma, St. Louis, MO, USA), the mouse serum was collected and purified the antibodies with protein G-agarose column (Pierce/Thermo Scientific, Waltham, Massachusetts, USA). The anti-rPoMPO Abs titer was tested by enzyme-linked immunosorbent assay, and then the specificity was analyzed using Western blotting and Mass spectrometry analysis (Sangon Biotech, Shanghai, China).
The concentration of rPoMPO was determined to be 1 mg mL-1 by using the Bradford kit (P0006C, Beyotime, Shanghai, China). BALB/c mice were immunized with 100 µg rPoMPO according to previously reported methods (29 (link)). After two boosters with a mixture of rPoMPO and incomplete Freund’s adjuvant (Sigma, St. Louis, MO, USA), the mouse serum was collected and purified the antibodies with protein G-agarose column (Pierce/Thermo Scientific, Waltham, Massachusetts, USA). The anti-rPoMPO Abs titer was tested by enzyme-linked immunosorbent assay, and then the specificity was analyzed using Western blotting and Mass spectrometry analysis (Sangon Biotech, Shanghai, China).
5
Recombinant Expression of Glycosyltransferases
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Expression of recombinant UGT1 and UGT2 were referred to the method described by Kim et al.29 (link) Briefly, the glycosyltransferase genes from Oryza sativa (UGT1, GenBank accession no. AK121682) and S. rebaudiana (UGT2, GenBank accession no. AY345974) were codon-optimized and synthesized by GenScript (Nanjing, China). Respectively, the resulting UGT1 or UGT2 was cloned in the pET28a(+) vector and transformed into the E. coli BL21(DE3) competent cells (TransGen Biotech, Beijing, China), resulting in the recombinant E. coli BL21 (pet28a-SrUGT76G1) strain or E. coli BL21 (pet28a-OsEUGT11) strain. The recombinant strain was cultivated in 400 mL Luria–Bertani (LB) medium containing kanamycin (50 mg mL−1) in 1 L flask until the OD600 reached approximately 0.6. The protein expression was induced by the addition of IPTG 0.1 mM IPTG and cells were incubated ∼14 hours at 18 °C. The cells were collected from the medium and resuspended in the Tris–HCl buffer at pH 7.0. Subsequently, the cells were lysed by ultra-sonication in an ice-water bath. After centrifugation, the His-tagged protein in the supernatant was purified through the affinity adsorption on Ni-NTA agarose column (Invitrogen). Protein mass concentration was measured by TaKaRa Bradford Protein Assay Kit with bovine serum albumin as a reference protein.
6
Cloning and Expression of SEQ ID NO: 3
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Example 2
The PCR product (SEQ ID NO: 3) was double-digested by restriction enzyme XhoI and HindIII and ligated with the vector pET24b plasmid (given by Hebei Chuangyue Company, Mr. Liu Jilai) digested by the same enzyme and transformed into E. coli BL21 (purchased from TransGen Biotech Company). The positive clones were analyzed by PCR and identified by restriction enzyme digestion and nucleotide sequencing. The results indicating that the sequence of SEQ ID NO: 3 was successfully inserted into the plasmid vector and successfully transformed into the E. coli BL21
7
Bacterial Strains for Protein Expression
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Escherichia coli DH5α and E. coli BL21 used for protein expression and purification were purchased from TransGen (Beijing, China) and cultured in LB medium. Wild-type L. monocytogenes strain EGD and its hly-deficient mutant EGDΔhly and the complementation strain EGDΔhly::hly were kind gifts from Pascale Cossart (Institut Pasteur, Paris, France) and were grown in Trypticase soy broth (TSB) medium with shaking at 37°C.
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8
Protein-protein Interaction Analysis
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The MdNAC4 and MdPYL4 CDSs were fused to the pET32a and pGEX-4 T-1 vectors, respectively, and these two constructed recombinant plasmids were transformed into E. coli BL21 (TransGen, Beijing, China). The induction of HIS- and GST-tagged protein expression was achieved using 1 mm isopropyl-β-D-thiogalactopyranoside. After the incubation of MdNAC4-HIS with MdPYL4-GST or GST, pull-down assays were performed using a HIS-tagged protein purification kit (CW Biotech, Taizhou, China). The eluted proteins were separated and detected by immunoblotting using anti-HIS and anti-GST antibodies (Abmart, Shanghai, China).
9
Recombinant Guanine Deaminase Enzyme
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The previous report showed guanine deaminase catalyzed the conversion of guanine to xanthine in wild-type Saccharomyces cerevisiae,18 (link) and GUD1 (YDL238c) encoded guanine deaminase.15 (link) The sequence of ScGUD1 (GUD1 from S. cerevisiae) was acquired from GenBank (accession no. NM_001180298). General DNA manipulation in S. cerevisiae was performed using standard methods.19 (link) Polymerase chain reaction (PCR)-specific primers with the BamHI restriction enzyme site
To obtain the recombinant pMAL-ScGUD1, we purified this amplified open reading frame (ORF) of ScGUD1, using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). Later, ScGUD1 was linked with pMAL-c5X vector. And the recombinant expression plasmid was constructed and transformed into the expression host bacteria E. coli (BL21) (TransGen Biotech) since the ligated cloned fragment had been confirmed.
To obtain the recombinant pMAL-ScGUD1, we purified this amplified open reading frame (ORF) of ScGUD1, using the AxyPrep DNA Gel Extraction Kit (Axygen, USA). Later, ScGUD1 was linked with pMAL-c5X vector. And the recombinant expression plasmid was constructed and transformed into the expression host bacteria E. coli (BL21) (TransGen Biotech) since the ligated cloned fragment had been confirmed.
10
Recombinant Rhodococcus sp. Glycanase Purification
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We obtained DNA polymerase, BamHI and HindIII restriction endonuclease, T4 ligase, TIAN amp Bacteria DNA Kit, TIAN prep Mini Plasmid Kit, Universal DNA Purification Kit, and EasyPure Quick Gel Extraction Kit., Ltd. from TRAN (Beijing, China). Ni-NTA agarose was obtained from Qiagen Gmbh (Hilden, Germany). Fluorogenic glycanase substrate of 4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotrioside (4-MUF-3-NAG) was obtained from Sigma-Aldrich, Co., (St. Louis, MO, United States). CFDA SE Cell Proliferation Fluorescent Probe was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Host cell strains, E. coli TOP10 and E. coli BL21, were obtained from TransGen Biotech CO., Ltd. (Shanghai, China) and the pET-30a (+) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals used in this study were of analytical grade. The Rhodococcus sp. (GX12401) was reserved in the China General Microbiological Culture Collection Center (CGMCC) under the number CGMCC NO.23759.
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