Structured Review

Merck KGaA bl21
Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) <t>BL21/pAK400</t> combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).
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Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

Journal: Scientific Reports

doi: 10.1038/s41598-017-03957-6

Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) BL21/pAK400 combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).
Figure Legend Snippet: Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) BL21/pAK400 combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).

Techniques Used:

Related Articles

Clone Assay:

Article Title: DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Article Snippet: Escherichia coli strain TOP-10 (Thermo Fisher Scientific Inc.; Waltham, MA) was used for cloning. .. E . coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: The primers used in the amplification and cloning steps were 5’-ggtgatgatgatgacaagaacgtgaaaaagttccctgaaggattcc-3’ and 5’-ggagatgggaagtcattatcagtcttccagaccgttgtttttaac-3’. .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Article Title: Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae
Article Snippet: .. Escherichia coli strains TOP-10 (Life Technologies Corp., Carlsbad, CA), Rosetta-gami 2, and BL21(DE3)(pLysS) (Merck KGaA, Darmstadt, Germany) were used for cloning and protein expression. .. GyrA and GyrB expression plasmids were constructed on the basis of pET-20b (+) (Merck KGaA).

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 synthetic DNA fragments were purchased from Integrated DNA Technologies (Coralville, IA) and cloned into a modified pET40 vector (fragment encoding DSBC signal peptide was erased from it by Protein Expression and Purification facility of the Hebrew University to allow cytoplasmic expression of DSBC). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Centrifugation:

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0). .. Following centrifugation at 5000g for 5 minutes, supernatant was removed and precipitate solved in 5ml of urea 8M.

Amplification:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: Each amplified fragment was digested with EcoRI and SalI and ligated into the pET41a(+) vector (Merck Millipore) digested with the same enzymes. .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore).

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: The primers used in the amplification and cloning steps were 5’-ggtgatgatgatgacaagaacgtgaaaaagttccctgaaggattcc-3’ and 5’-ggagatgggaagtcattatcagtcttccagaccgttgtttttaac-3’. .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: The fragment corresponding to the first domain of NEP3 between the Lys-Arg cleavage sites was amplified by PCR, cloned and expressed as a His6 -thioredoxin fusion protein in Shuffle T7 Escherichia coli strain (New England Biolabs). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Binding Assay:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: Paragraph title: Chitin binding assay ... The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore).

Construct:

Article Title: Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae
Article Snippet: Escherichia coli strains TOP-10 (Life Technologies Corp., Carlsbad, CA), Rosetta-gami 2, and BL21(DE3)(pLysS) (Merck KGaA, Darmstadt, Germany) were used for cloning and protein expression. .. GyrA and GyrB expression plasmids were constructed on the basis of pET-20b (+) (Merck KGaA).

Incubation:

Article Title: Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation
Article Snippet: BL21(DE3) cells (Merck Biosciences) were transformed with plasmids pET156-UBE2L3*-TAG3 and pCDF-PylST (plasmid harbouring constitutive copies of the Mb PylRS/tRNACUA pair, a kind gift from Dr. Jason Chin) and used to inoculate 200 mL LB media containing ampicillin (100 μg mL-1 ) and spectinomycin (50 μg mL-1 ). .. After overnight incubation at 37 C, 6 × 1 L of LB medium containing ampicillin (100 μg mL-1 ) and spectinomycin (25 μg mL-1 ) were each inoculated with 30 mL overnight culture and incubated at 37 °C, 200 rpm, until cell density reached OD600 = 0.8-0.9.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6. .. The resuspended cells were incubated on ice during the sonication procedure, and a 3 min cooling step was introduced between the ultrasound pulses.

Expressing:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore). .. Protein expression was induced with IPTG according to the manufacturer’s instruction.

Article Title: Identification of new FGF1 binding partners—Implications for its intracellular function
Article Snippet: .. Protein Expression and Purification Recombinant FGF1, SBP‐FGF1, and C‐terminal fragment of nucleolin (residues 284–707) with GST tag were produced in E. coli Bl21(DE3)pLysS or Bl21(DE3)‐RIL strains (from Merck, Germany). .. SBP‐FGF1 was purified on Heparin‐Sepharose CL‐6B column.

Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
Article Snippet: Vectors, bacterial strains, enzymes, reagents and sequencing Phagemid vector pEB32x was used for phage display and vectors pAK400 and pLK04 for soluble expression. .. E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F ’ proAB lacI q ZΔM15 Tn10 (Tet r )]) was purchased from Stratagene (LaJolla, USA) and BL21 (B F − ompT gal dcm lon hsdS B (r B − m B − ) [malB + ]K −12 (λ S )) was purchased from Merck (Darmstadt, Germany).

Article Title: DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Article Snippet: .. E . coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. .. The plasmid vector pET-20b(+) (Merck KGaA) was used for the construction of expression plasmids.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Paragraph title: Expression and purification of the wild-type and mutant bglTm proteins ... BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Article Title: Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae
Article Snippet: .. Escherichia coli strains TOP-10 (Life Technologies Corp., Carlsbad, CA), Rosetta-gami 2, and BL21(DE3)(pLysS) (Merck KGaA, Darmstadt, Germany) were used for cloning and protein expression. .. GyrA and GyrB expression plasmids were constructed on the basis of pET-20b (+) (Merck KGaA).

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Paragraph title: Recombinant expression and purification of toxins ... Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: Paragraph title: Recombinant protein expression and purification ... Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0).

Modification:

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 synthetic DNA fragments were purchased from Integrated DNA Technologies (Coralville, IA) and cloned into a modified pET40 vector (fragment encoding DSBC signal peptide was erased from it by Protein Expression and Purification facility of the Hebrew University to allow cytoplasmic expression of DSBC). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Transformation Assay:

Article Title: Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation
Article Snippet: .. BL21(DE3) cells (Merck Biosciences) were transformed with plasmids pET156-UBE2L3*-TAG3 and pCDF-PylST (plasmid harbouring constitutive copies of the Mb PylRS/tRNACUA pair, a kind gift from Dr. Jason Chin) and used to inoculate 200 mL LB media containing ampicillin (100 μg mL-1 ) and spectinomycin (50 μg mL-1 ). .. After overnight incubation at 37 C, 6 × 1 L of LB medium containing ampicillin (100 μg mL-1 ) and spectinomycin (25 μg mL-1 ) were each inoculated with 30 mL overnight culture and incubated at 37 °C, 200 rpm, until cell density reached OD600 = 0.8-0.9.

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore). .. Protein expression was induced with IPTG according to the manufacturer’s instruction.

Article Title: The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif
Article Snippet: For plasmid manipulation and protein overproduction we used Escherichia coli strains XL1blue (Bullock et al., ) and BL21(DE3) pLysS (Merck Millipore), respectively. .. For mating experiments we used S. lividans strains TK64 and TK54 (Hopwood et al., ) transformed with the required plasmids.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6. .. Then, protein expression was induced with 1 mM isopropyl β-thio-galactopyranoside (IPTG) for 24 h at 20°C with shaking at 150 rpm.

Over Expression:

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: .. Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0). .. Following centrifugation at 5000g for 5 minutes, supernatant was removed and precipitate solved in 5ml of urea 8M.

Flow Cytometry:

Article Title: Identification of new FGF1 binding partners—Implications for its intracellular function
Article Snippet: Protein Expression and Purification Recombinant FGF1, SBP‐FGF1, and C‐terminal fragment of nucleolin (residues 284–707) with GST tag were produced in E. coli Bl21(DE3)pLysS or Bl21(DE3)‐RIL strains (from Merck, Germany). .. GST‐nucleolin was expressed and purified using Glutathione Sepharose 4 Fast Flow column as described previously .

Sequencing:

Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
Article Snippet: Paragraph title: Vectors, bacterial strains, enzymes, reagents and sequencing ... E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F ’ proAB lacI q ZΔM15 Tn10 (Tet r )]) was purchased from Stratagene (LaJolla, USA) and BL21 (B F − ompT gal dcm lon hsdS B (r B − m B − ) [malB + ]K −12 (λ S )) was purchased from Merck (Darmstadt, Germany).

Chromatography:

Article Title: Lattice Shrinkage by Incorporation of Recombinant Starmaker‐Like Protein within Bioinspired Calcium Carbonate Crystals
Article Snippet: .. Briefly, Stm‐l was overexpressed in BL21(DE3)pLysS Escherichia coli cells (Merck KGaA, Darmstadt, Germany) and purified through fractionation with ammonium sulfate, size‐exclusion chromatography, and anion‐exchange chromatography. ..

Polymerase Chain Reaction:

Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
Article Snippet: E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F ’ proAB lacI q ZΔM15 Tn10 (Tet r )]) was purchased from Stratagene (LaJolla, USA) and BL21 (B F − ompT gal dcm lon hsdS B (r B − m B − ) [malB + ]K −12 (λ S )) was purchased from Merck (Darmstadt, Germany). .. Minipreps, gel extractions and PCR purifications were done with Thermo Scientific kits (Waltham, USA).

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: The fragment corresponding to the first domain of NEP3 between the Lys-Arg cleavage sites was amplified by PCR, cloned and expressed as a His6 -thioredoxin fusion protein in Shuffle T7 Escherichia coli strain (New England Biolabs). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Sonication:

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6. .. The induced bacteria were resuspended in 5 mL of 10 mM sodium phosphate buffer, pH 7, containing 100 mM NaCl and 20 mM imidazole and then disrupted by sonication (4 ultrasound pulses of 15 s at output 3 in a Branson Sonifier 250 (Branson Instruments, Stanford, CT, US)).

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: .. Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0). .. Following centrifugation at 5000g for 5 minutes, supernatant was removed and precipitate solved in 5ml of urea 8M.

Recombinant:

Article Title: Identification of new FGF1 binding partners—Implications for its intracellular function
Article Snippet: .. Protein Expression and Purification Recombinant FGF1, SBP‐FGF1, and C‐terminal fragment of nucleolin (residues 284–707) with GST tag were produced in E. coli Bl21(DE3)pLysS or Bl21(DE3)‐RIL strains (from Merck, Germany). .. SBP‐FGF1 was purified on Heparin‐Sepharose CL‐6B column.

Article Title: Lattice Shrinkage by Incorporation of Recombinant Starmaker‐Like Protein within Bioinspired Calcium Carbonate Crystals
Article Snippet: Protein preparation Recombinant, nontagged Stm‐l protein was obtained as previously described. .. Briefly, Stm‐l was overexpressed in BL21(DE3)pLysS Escherichia coli cells (Merck KGaA, Darmstadt, Germany) and purified through fractionation with ammonium sulfate, size‐exclusion chromatography, and anion‐exchange chromatography.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6. .. Then, protein expression was induced with 1 mM isopropyl β-thio-galactopyranoside (IPTG) for 24 h at 20°C with shaking at 150 rpm.

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Paragraph title: Recombinant expression and purification of toxins ... Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: Paragraph title: Recombinant protein expression and purification ... Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0).

DNA Cleavage Assay:

Article Title: DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Article Snippet: E . coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. .. Relaxed and supercoiled pBR322 DNA (John Innes Enterprises Ltd.; Norwich, United Kingdom) were used for the DNA supercoiling assay and DNA cleavage assay.

Magnetic Beads:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore). .. Purified proteins were desalted using Microcon Centrifugal Filters (Merck Millipore), and were applied to Chitin Magnetic Beads (New England Biolabs) according to the manufacturer’s protocol.

Mutagenesis:

Article Title: Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation
Article Snippet: This yielded the plasmid pET156-UBE2L3*-TAG3 (* corresponds to a C17S and C137S mutant). .. BL21(DE3) cells (Merck Biosciences) were transformed with plasmids pET156-UBE2L3*-TAG3 and pCDF-PylST (plasmid harbouring constitutive copies of the Mb PylRS/tRNACUA pair, a kind gift from Dr. Jason Chin) and used to inoculate 200 mL LB media containing ampicillin (100 μg mL-1 ) and spectinomycin (50 μg mL-1 ).

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Paragraph title: Expression and purification of the wild-type and mutant bglTm proteins ... BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Size-exclusion Chromatography:

Article Title: Lattice Shrinkage by Incorporation of Recombinant Starmaker‐Like Protein within Bioinspired Calcium Carbonate Crystals
Article Snippet: .. Briefly, Stm‐l was overexpressed in BL21(DE3)pLysS Escherichia coli cells (Merck KGaA, Darmstadt, Germany) and purified through fractionation with ammonium sulfate, size‐exclusion chromatography, and anion‐exchange chromatography. ..

Purification:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore). .. Purified proteins were desalted using Microcon Centrifugal Filters (Merck Millipore), and were applied to Chitin Magnetic Beads (New England Biolabs) according to the manufacturer’s protocol.

Article Title: Identification of new FGF1 binding partners—Implications for its intracellular function
Article Snippet: .. Protein Expression and Purification Recombinant FGF1, SBP‐FGF1, and C‐terminal fragment of nucleolin (residues 284–707) with GST tag were produced in E. coli Bl21(DE3)pLysS or Bl21(DE3)‐RIL strains (from Merck, Germany). .. SBP‐FGF1 was purified on Heparin‐Sepharose CL‐6B column.

Article Title: Lattice Shrinkage by Incorporation of Recombinant Starmaker‐Like Protein within Bioinspired Calcium Carbonate Crystals
Article Snippet: .. Briefly, Stm‐l was overexpressed in BL21(DE3)pLysS Escherichia coli cells (Merck KGaA, Darmstadt, Germany) and purified through fractionation with ammonium sulfate, size‐exclusion chromatography, and anion‐exchange chromatography. ..

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Paragraph title: Expression and purification of the wild-type and mutant bglTm proteins ... BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Paragraph title: Recombinant expression and purification of toxins ... Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: Paragraph title: Recombinant protein expression and purification ... Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0).

Protein Purification:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore). .. Protein purification using glutathione beads was done using MagneGST Protein Purification System (Promega).

Transgenic Assay:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: Chitin binding assay Using full-length obst-E-a and -b cDNAs (see “Transgenic flies”) as templates, the coding sequences excluding the predicted N-terminal signal peptides were amplified using the following primers: a Fw, 5’-AGTCAGGAATTCTTTGGCTCAATGGCTGCC-3’; a Rv, 5’- AGTCGTCGACGTTCTTCCTGGCGTGAAG-3’; b Fw, 5’- TCAGTCAGTCGAATTCTTTGGCTCAATGGCTCTTGGC-3’ and b Rv, 5’- TCAGTCAGTCGTCGACATAGTCCTCCGGCTGA-3’. .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore).

Affinity Chromatography:

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: Purification of recombinant protein was performed using affinity chromatography under denaturing condition. .. Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0).

Fast Protein Liquid Chromatography:

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC. .. The polyhistidine tag of the fusion proteins was used for purification from the E. coli lysate by nickel affinity FPLC.

IA:

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 synthetic DNA fragments were purchased from Integrated DNA Technologies (Coralville, IA) and cloned into a modified pET40 vector (fragment encoding DSBC signal peptide was erased from it by Protein Expression and Purification facility of the Hebrew University to allow cytoplasmic expression of DSBC). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Plasmid Preparation:

Article Title: Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation
Article Snippet: .. BL21(DE3) cells (Merck Biosciences) were transformed with plasmids pET156-UBE2L3*-TAG3 and pCDF-PylST (plasmid harbouring constitutive copies of the Mb PylRS/tRNACUA pair, a kind gift from Dr. Jason Chin) and used to inoculate 200 mL LB media containing ampicillin (100 μg mL-1 ) and spectinomycin (50 μg mL-1 ). .. After overnight incubation at 37 C, 6 × 1 L of LB medium containing ampicillin (100 μg mL-1 ) and spectinomycin (25 μg mL-1 ) were each inoculated with 30 mL overnight culture and incubated at 37 °C, 200 rpm, until cell density reached OD600 = 0.8-0.9.

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: Each amplified fragment was digested with EcoRI and SalI and ligated into the pET41a(+) vector (Merck Millipore) digested with the same enzymes. .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore).

Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli
Article Snippet: Vector pUC57 was obtained from Genscript (Piscataway Township, USA). .. E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F ’ proAB lacI q ZΔM15 Tn10 (Tet r )]) was purchased from Stratagene (LaJolla, USA) and BL21 (B F − ompT gal dcm lon hsdS B (r B − m B − ) [malB + ]K −12 (λ S )) was purchased from Merck (Darmstadt, Germany).

Article Title: The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif
Article Snippet: .. For plasmid manipulation and protein overproduction we used Escherichia coli strains XL1blue (Bullock et al., ) and BL21(DE3) pLysS (Merck Millipore), respectively. .. For mating experiments we used S. lividans strains TK64 and TK54 (Hopwood et al., ) transformed with the required plasmids.

Article Title: DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Article Snippet: E . coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. .. The plasmid vector pET-20b(+) (Merck KGaA) was used for the construction of expression plasmids.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Expression and purification of the wild-type and mutant bglTm proteins The DNA segments encoding the wild-type and mutant bglTm proteins were produced by GenScript (Piscataway, NJ, US) and cloned into the pLATE51 expression vector (Thermo Scientific, Waltham, MA, US). .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 synthetic DNA fragments were purchased from Integrated DNA Technologies (Coralville, IA) and cloned into a modified pET40 vector (fragment encoding DSBC signal peptide was erased from it by Protein Expression and Purification facility of the Hebrew University to allow cytoplasmic expression of DSBC). .. Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC.

Negative Control:

Article Title: Mechanical Control of Whole Body Shape by a Single Cuticular Protein Obstructor-E in Drosophila melanogaster
Article Snippet: For negative control, pET41a(+) was digested with BamHI and BglII and self-ligated. .. The resultant expression vectors, pET41a-GST-His-Obst-E-a-His, pET41a-GST-His-Obst-E-b-His and pET41a-GST-His-linker-His were transformed into BL21(DE3)pLysS strain (Merck Millipore).

Positron Emission Tomography:

Article Title: DC-159a Shows Inhibitory Activity against DNA Gyrases of Mycobacterium leprae
Article Snippet: E . coli strains Rosetta-gami 2(DE3)pLysS and BL21(DE3)pLysS (Merck KGaA, Darmstadt, Germany) were used for protein expression. .. The plasmid vector pET-20b(+) (Merck KGaA) was used for the construction of expression plasmids.

Article Title: Amino Acid Substitutions at Position 95 in GyrA Can Add Fluoroquinolone Resistance to Mycobacterium leprae
Article Snippet: Escherichia coli strains TOP-10 (Life Technologies Corp., Carlsbad, CA), Rosetta-gami 2, and BL21(DE3)(pLysS) (Merck KGaA, Darmstadt, Germany) were used for cloning and protein expression. .. GyrA and GyrB expression plasmids were constructed on the basis of pET-20b (+) (Merck KGaA).

Produced:

Article Title: Identification of new FGF1 binding partners—Implications for its intracellular function
Article Snippet: .. Protein Expression and Purification Recombinant FGF1, SBP‐FGF1, and C‐terminal fragment of nucleolin (residues 284–707) with GST tag were produced in E. coli Bl21(DE3)pLysS or Bl21(DE3)‐RIL strains (from Merck, Germany). .. SBP‐FGF1 was purified on Heparin‐Sepharose CL‐6B column.

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Expression and purification of the wild-type and mutant bglTm proteins The DNA segments encoding the wild-type and mutant bglTm proteins were produced by GenScript (Piscataway, NJ, US) and cloned into the pLATE51 expression vector (Thermo Scientific, Waltham, MA, US). .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

Concentration Assay:

Article Title: Probes of Ubiquitin E3 ligases distinguish different stages of Parkin activation
Article Snippet: BL21(DE3) cells (Merck Biosciences) were transformed with plasmids pET156-UBE2L3*-TAG3 and pCDF-PylST (plasmid harbouring constitutive copies of the Mb PylRS/tRNACUA pair, a kind gift from Dr. Jason Chin) and used to inoculate 200 mL LB media containing ampicillin (100 μg mL-1 ) and spectinomycin (50 μg mL-1 ). .. Propargyloxycarbonyl-L-lysine (ProcK; Iris Biotech GmbH, #HAA2095) (300 mM stock solution in H2 O and pH adjusted to ~7) was then added to a final concentration of 3 mM.

Article Title: Dynamics of venom composition across a complex life cycle
Article Snippet: Nv1 and NvePTx1 were expressed in BL21(DE3) E. coli (Merck Millipore) strain as fusions with His6 -DSBC. .. The recombinant toxins were then purified by reverse phase FPLC on a Resource RPC column (GE Healthcare) using an acetonitrile concentration gradient in 0.1% trifluoroacetic acid.

Article Title: Fibrinogen and Fibronectin Binding Activity and Immunogenic Nature of Choline Binding Protein M
Article Snippet: When OD600 reached 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma-Aldric, USA) was added to a final concentration of 1mM, and the cells were grown for 4 hours at 37 °C with continuous shaking. .. Briefly, after overexpression of BL21 (DE3) in 250 ml LB broth (Merck, Germany) containing ampicillin, the bacterial culture was centrifuged at 5000g for 5 minutes and plate lysed by sonication on ice in solution buffer (300 mM NaCl, 50 mM NaH2PO4, pH:8.0).

Fractionation:

Article Title: Lattice Shrinkage by Incorporation of Recombinant Starmaker‐Like Protein within Bioinspired Calcium Carbonate Crystals
Article Snippet: .. Briefly, Stm‐l was overexpressed in BL21(DE3)pLysS Escherichia coli cells (Merck KGaA, Darmstadt, Germany) and purified through fractionation with ammonium sulfate, size‐exclusion chromatography, and anion‐exchange chromatography. ..

DNA Purification:

Article Title: Search for independent (β/α)4 subdomains in a (β/α)8 barrel β-glucosidase
Article Snippet: Recombinant pLATE51 vectors encoding bglTm were propagated in XL1 Blue cells (Agilent, Santa Clara, CA, US) and extracted with a Wizard Plus SV Miniprep DNA Purification System (Promega, Madison, WI, US). .. BL21(DE3) cells (Merck Millipore, Billerica, MA, US) transformed with the recombinant pLATE51 vectors were cultivated in Luria broth (500 mL) containing ampicillin (50 μg/mL) at 37°C with shaking at 150 rpm until the culture reached an optical density at 600 nm in the range 0.4–0.6.

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  • 93
    Merck KGaA e coli bl21
    SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in <t>BL21</t> E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands
    E Coli Bl21, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA bl21 de3 plyss strain
    Small Scale expression analysis from pBDDP-SPR3 constructs. GTPases were expressed in the <t>Bl21</t> (DE3) <t>pLysS</t> strain of E. coli in Luria Bertani broth supplemented with 30 μg/ml kanamycin sulfate. A 1 ml sample of cells was collected before induction and at the time of cell harvesting. This allowed the assessment of total cell protein by SDS-PAGE, and the visualisation of over-expressed target protein. A 40 ml sample of expression culture was applied to a small scale CoNTA IMAC purification to demonstrate the presence of soluble his-tagged protein. (As reported by others, CoNTA spin column purification has proved particularly useful in this respect and gives a consistently clear indication of levels and integrity of recombinant proteins [20] .) In all three cases there was significant soluble expression of the GTPase allowing progression to FPLC scale purification (H 8 -H 8 -KRas 1–169, 24.8 kDa; H 8 -H 8 -Rac1-2-177, 26.2 kDa; H 8 -H 8 -RalB, 25.5 kDa).
    Bl21 De3 Plyss Strain, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA bl21
    Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) <t>BL21/pAK400</t> combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).
    Bl21, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    bl21 - by Bioz Stars, 2020-04
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    Image Search Results


    SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands

    Journal: Advanced Biomedical Research

    Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications

    doi: 10.4103/2277-9175.161576

    Figure Lengend Snippet: SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands

    Article Snippet: E. coli BL21 (DE3) and pET-24a(+) plasmid (Merck KGaA, Darmstadt, Germany) were used as expression host and vector, respectively.

    Techniques: SDS Page, Western Blot, Recombinant, Plasmid Preparation, Concentration Assay, Molecular Weight, Marker, Expressing

    SDS-PAGE and western blot Analysis of the purified recombinant protein after purification: (A) Lane 1, purified protein; 2; Lane M, molecular weight marker; Lane 3, bacterial lysate after 3h incubation. (B) Lane 1, purified protein; Lane M, molecular weight marker; Lane 2, BL21 cell lysate.

    Journal: Iranian Journal of Microbiology

    Article Title: Cloning, expression and purification of the factor H binding protein and its interaction with factor H

    doi:

    Figure Lengend Snippet: SDS-PAGE and western blot Analysis of the purified recombinant protein after purification: (A) Lane 1, purified protein; 2; Lane M, molecular weight marker; Lane 3, bacterial lysate after 3h incubation. (B) Lane 1, purified protein; Lane M, molecular weight marker; Lane 2, BL21 cell lysate.

    Article Snippet: E. coli BL21 (DE3) was used for protein expression as host with 50μg/μl kanamycin (Merck,Germany) in LB medium for selection, 0.5 mM IPTG (Isopropyl-beta-D-thiogalacto-pyranoside) (Merck,Germany) as inducer.

    Techniques: SDS Page, Western Blot, Purification, Recombinant, Molecular Weight, Marker, Incubation

    Small Scale expression analysis from pBDDP-SPR3 constructs. GTPases were expressed in the Bl21 (DE3) pLysS strain of E. coli in Luria Bertani broth supplemented with 30 μg/ml kanamycin sulfate. A 1 ml sample of cells was collected before induction and at the time of cell harvesting. This allowed the assessment of total cell protein by SDS-PAGE, and the visualisation of over-expressed target protein. A 40 ml sample of expression culture was applied to a small scale CoNTA IMAC purification to demonstrate the presence of soluble his-tagged protein. (As reported by others, CoNTA spin column purification has proved particularly useful in this respect and gives a consistently clear indication of levels and integrity of recombinant proteins [20] .) In all three cases there was significant soluble expression of the GTPase allowing progression to FPLC scale purification (H 8 -H 8 -KRas 1–169, 24.8 kDa; H 8 -H 8 -Rac1-2-177, 26.2 kDa; H 8 -H 8 -RalB, 25.5 kDa).

    Journal: Protein Expression and Purification

    Article Title: A fully automated procedure for the parallel, multidimensional purification and nucleotide loading of the human GTPases KRas, Rac1 and RalB

    doi: 10.1016/j.pep.2017.01.010

    Figure Lengend Snippet: Small Scale expression analysis from pBDDP-SPR3 constructs. GTPases were expressed in the Bl21 (DE3) pLysS strain of E. coli in Luria Bertani broth supplemented with 30 μg/ml kanamycin sulfate. A 1 ml sample of cells was collected before induction and at the time of cell harvesting. This allowed the assessment of total cell protein by SDS-PAGE, and the visualisation of over-expressed target protein. A 40 ml sample of expression culture was applied to a small scale CoNTA IMAC purification to demonstrate the presence of soluble his-tagged protein. (As reported by others, CoNTA spin column purification has proved particularly useful in this respect and gives a consistently clear indication of levels and integrity of recombinant proteins [20] .) In all three cases there was significant soluble expression of the GTPase allowing progression to FPLC scale purification (H 8 -H 8 -KRas 1–169, 24.8 kDa; H 8 -H 8 -Rac1-2-177, 26.2 kDa; H 8 -H 8 -RalB, 25.5 kDa).

    Article Snippet: 2.2 Expression of the CDC25 GEF domain of SOS1 and G-domains of KRas 4B, Rac1 and RalB in pBDDP-SPR3 Plasmids were transformed into the BL21 (DE3) pLysS strain of E. coli (Merck Millipore) and the transformed cells were cultured overnight at 37 °C in Luria Bertani broth supplemented with 30 μg/ml kanamycin sulfate (Melford Laboratories Ltd).

    Techniques: Expressing, Construct, Cell Harvesting, SDS Page, Purification, Recombinant, Fast Protein Liquid Chromatography

    Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) BL21/pAK400 combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).

    Journal: Scientific Reports

    Article Title: Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli

    doi: 10.1038/s41598-017-03957-6

    Figure Lengend Snippet: Correlation between mRNA minimum free energy (ΔG) and protein yield. ( a ) BL21/pAK400 combination. ( b ) BL21/pLK04 combination. ( c ) XL1-Blue/pAK400 combination. ( d ) XL1-Blue/pLK04 combination. Data points are marked as follows: the parent Fab0 (sphere) and the variants 1 (diamond), 2 (line), 3 (triangle) and 4 (square).

    Article Snippet: E. coli XL-1 Blue (recA1 endA1 gryA96 thi-1 hsdR17 supE44 relA1 lac [F ’ proAB lacI q ZΔM15 Tn10 (Tet r )]) was purchased from Stratagene (LaJolla, USA) and BL21 (B F − ompT gal dcm lon hsdS B (r B − m B − ) [malB + ]K −12 (λ S )) was purchased from Merck (Darmstadt, Germany).

    Techniques: