Plasmid pKW01 (Cas1-WT) was constructed by through amplification of pWJ40 as a template for polymerase chain reactions (PCRs) to clone Cas1 into pET28b-His10Smt3 using the primers PS192 and PS193. Full sequencing of cloned DNA fragment confirmed perfect matches to the original sequence. The pKW01 plasmid was transformed into E. coli BL21 (DE3) Rosetta 2 cells (EMD Millipore). Cultures were grown and protein was purified by Ni- affinity chromatography step, as mentioned before in Cas9 purification. The 200 mM imidazole elutes containing the His10-Smt3 tagged Cas1 polypeptide was pooled together. The His10-Smt3 affinity tag was removed by cleavage with SUMO protease during overnight dialysis against 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 20 mM and 10% glycerol. The tagless Cas1 protein was separated from the fusion tag by using a second Ni-NTA affinity step. The protein was further purified by size exclusion chromatography using a Superdex 200 10/300 GL in 20 mM Tris HCl pH 7.5, 500 mM KCl, 1 mM TCEP, and 5% glycerol. The elution peak from the size exclusion was aliquoted, frozen and kept at −80 °C.
E coli bl21 de3 rosetta 2 cells
Manufactured by Merck Group
E. coli BL21 (DE3) Rosetta 2 cells are a bacterial strain commonly used in molecular biology and protein expression applications. They are derived from the E. coli BL21 (DE3) strain and are engineered to enhance the expression of proteins from organisms with codon usage that differs from standard E. coli. The Rosetta 2 cells provide additional tRNAs to improve the translation of mRNA sequences that contain rare codons.
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Lab products found in correlation
3 protocols using e coli bl21 de3 rosetta 2 cells
1
Cloning and Purification of Cas1 Protein
2
Cloning and Purification of Cas1 Protein
3
Recombinant PbMCS Enzyme Production
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The sequence of the mature PbMCS without mitochondrial import peptide was used for production of the recombinant enzyme in Escherichia coli. The coding sequence was amplified from cDNA of P. lutzii isolate Pb01 and cloned into a modified pET43 plasmid in frame with a His-Tag sequence (Hortschansky et al. 2007 (link)). The plasmid was transferred into E. coli BL21 (DE3) Rosetta 2 cells (Novagen/Merck), which were used for overproduction of the recombinant enzyme. Protein production was induced by cultivation in Overnight Express Instant TB medium at 30 °C. Cells were collected by centrifugation and resuspended in buffer A (50 mM Tris/HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and disrupted by sonication. Lysates were clarified by centrifugation and loaded onto a nickel-Sepharose 6 Fast Flow gravity-flow column (GE Healthcare). After six washes with buffer A containing 30 mM imidazole the protein was eluted in buffer A containing 200 mM imidazole. The recombinant protein purity was assessed by SDS-PAGE. Fractions with purified enzyme were combined and desalted using centrifugal filter devices (Merck). Then, glycerol was added to purified MCS and samples were stored at − 20 °C. Protein concentrations were determined by using the Bradford protein assay kit from Bio-Rad and bovine serum albumin was employed as standard.
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