strain bl21∷de3  (Millipore)


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    Millipore strain bl21∷de3
    Strain Bl21∷De3, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/strain bl21∷de3/product/Millipore
    Average 81 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    strain bl21∷de3 - by Bioz Stars, 2020-04
    81/100 stars

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    Plasmid Preparation:

    Article Title: Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis
    Article Snippet: .. Escherichia coli strains DH5α and DH10b (Bethesda Research Laboratories) were employed for plasmid construction, and strain BL21∷DE3 (Novagen) was used for protein expression. .. Standard procedures were followed for DNA manipulations and sequence analysis ( ).

    Expressing:

    Article Title: Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis
    Article Snippet: .. Escherichia coli strains DH5α and DH10b (Bethesda Research Laboratories) were employed for plasmid construction, and strain BL21∷DE3 (Novagen) was used for protein expression. .. Standard procedures were followed for DNA manipulations and sequence analysis ( ).

    Cell Culture:

    Article Title: Identification of a cAMP-dependent protein kinase catalytic subunit required for virulence and morphogenesis in Ustilago maydis
    Article Snippet: Cell culture, mating tests, and plant inoculations were performed as previously described ( ). .. Escherichia coli strains DH5α and DH10b (Bethesda Research Laboratories) were employed for plasmid construction, and strain BL21∷DE3 (Novagen) was used for protein expression.

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    Millipore e coli strain bl21
    Zymographic detection of the autolytic activity in recombinant His-tagged Atl. Protein extracts from E. coli <t>BL21</t> cells grown with IPTG were used in this study. The protein extract was separated by 12.5% SDS-gel electrophoresis. The SDS gel was impregnated with autoclaved S. aureus RN450 cells. Autolytic activity was detected by renaturing the autolysins by incubation of the gel in 50 mM Tri-HCl buffer, pH 7.5, containing 0.05% Triton X-100. Lane 1: full length His-tagged Atl; lane 2: Atl-1; lane 3: Atl-2; lane 4: Atl-3; lane 5: Atl-4; lane 6: Atl-5; lane 7: Atl-6; lane 8: Atl-7; lane 9: Atl-8.
    E Coli Strain Bl21, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli strain bl21/product/Millipore
    Average 99 stars, based on 284 article reviews
    Price from $9.99 to $1999.99
    e coli strain bl21 - by Bioz Stars, 2020-04
    99/100 stars
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    Zymographic detection of the autolytic activity in recombinant His-tagged Atl. Protein extracts from E. coli BL21 cells grown with IPTG were used in this study. The protein extract was separated by 12.5% SDS-gel electrophoresis. The SDS gel was impregnated with autoclaved S. aureus RN450 cells. Autolytic activity was detected by renaturing the autolysins by incubation of the gel in 50 mM Tri-HCl buffer, pH 7.5, containing 0.05% Triton X-100. Lane 1: full length His-tagged Atl; lane 2: Atl-1; lane 3: Atl-2; lane 4: Atl-3; lane 5: Atl-4; lane 6: Atl-5; lane 7: Atl-6; lane 8: Atl-7; lane 9: Atl-8.

    Journal: International Journal of Microbiology

    Article Title: High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    doi: 10.1155/2014/615965

    Figure Lengend Snippet: Zymographic detection of the autolytic activity in recombinant His-tagged Atl. Protein extracts from E. coli BL21 cells grown with IPTG were used in this study. The protein extract was separated by 12.5% SDS-gel electrophoresis. The SDS gel was impregnated with autoclaved S. aureus RN450 cells. Autolytic activity was detected by renaturing the autolysins by incubation of the gel in 50 mM Tri-HCl buffer, pH 7.5, containing 0.05% Triton X-100. Lane 1: full length His-tagged Atl; lane 2: Atl-1; lane 3: Atl-2; lane 4: Atl-3; lane 5: Atl-4; lane 6: Atl-5; lane 7: Atl-6; lane 8: Atl-7; lane 9: Atl-8.

    Article Snippet: In addition, E. coli strain BL21 (EMD Millipore) was used for all protein expression studies.

    Techniques: Activity Assay, Recombinant, SDS-Gel, Electrophoresis, Incubation

    Coomassie stained gels showing purity of recombinant His-tagged Atl. Recombinant Atl and Atl-1 proteins were overproduced in E. coli and purified as described in the Materials and Methods section. Lane M: standard protein markers; lanes 1 and 2: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl and grown without and with IPTG, respectively; lane 3: purified His-tagged Atl; lanes 4 and 5: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl1 and grown without and with IPTG, respectively; lane 6: purified His-tagged Atl-1.

    Journal: International Journal of Microbiology

    Article Title: High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    doi: 10.1155/2014/615965

    Figure Lengend Snippet: Coomassie stained gels showing purity of recombinant His-tagged Atl. Recombinant Atl and Atl-1 proteins were overproduced in E. coli and purified as described in the Materials and Methods section. Lane M: standard protein markers; lanes 1 and 2: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl and grown without and with IPTG, respectively; lane 3: purified His-tagged Atl; lanes 4 and 5: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl1 and grown without and with IPTG, respectively; lane 6: purified His-tagged Atl-1.

    Article Snippet: In addition, E. coli strain BL21 (EMD Millipore) was used for all protein expression studies.

    Techniques: Staining, Recombinant, Purification, Transformation Assay

    Zymographic detection of the autolytic activity in purified recombinant His-tagged Atl. Lane 1: total protein extract from S. aureus cells. Lanes 2 and 4: purified His-tagged Atl and Atl-1, respectively; lanes 3 and 5: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl or pRSETA- atl1 and grown with IPTG, respectively.

    Journal: International Journal of Microbiology

    Article Title: High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    doi: 10.1155/2014/615965

    Figure Lengend Snippet: Zymographic detection of the autolytic activity in purified recombinant His-tagged Atl. Lane 1: total protein extract from S. aureus cells. Lanes 2 and 4: purified His-tagged Atl and Atl-1, respectively; lanes 3 and 5: protein extract from E. coli BL21 cells transformed with plasmids pRSETA- atl or pRSETA- atl1 and grown with IPTG, respectively.

    Article Snippet: In addition, E. coli strain BL21 (EMD Millipore) was used for all protein expression studies.

    Techniques: Activity Assay, Purification, Recombinant, Transformation Assay

    Coomassie stained gels demonstrating overexpression of recombinant Atl. The 12.5% SDS-PAGE contains protein extracts of E. coli BL21 cells with pRSETA plasmids expressing either full length or truncated Atl proteins. The odd number labels are cells grown without IPTG and the even number labels are the cells grown with IPTG. Lane M: standard protein markers; lanes 1 and 2 - Atl (full length His-tagged Atl); lanes 3 and 4: Atl-1; lanes 5 and 6: Atl-2; lanes 7 and 8: Atl-3; lanes 9 and 10: Atl-4; lanes 11 and 12: Atl-6; lanes 13 and 14: Atl-5; lanes 15 and 16: Atl-7; lanes 17 and 18: Atl-8. The Atl number suffixes are indicated in Table 1 .

    Journal: International Journal of Microbiology

    Article Title: High Level Expression and Purification of Atl, the Major Autolytic Protein of Staphylococcus aureus

    doi: 10.1155/2014/615965

    Figure Lengend Snippet: Coomassie stained gels demonstrating overexpression of recombinant Atl. The 12.5% SDS-PAGE contains protein extracts of E. coli BL21 cells with pRSETA plasmids expressing either full length or truncated Atl proteins. The odd number labels are cells grown without IPTG and the even number labels are the cells grown with IPTG. Lane M: standard protein markers; lanes 1 and 2 - Atl (full length His-tagged Atl); lanes 3 and 4: Atl-1; lanes 5 and 6: Atl-2; lanes 7 and 8: Atl-3; lanes 9 and 10: Atl-4; lanes 11 and 12: Atl-6; lanes 13 and 14: Atl-5; lanes 15 and 16: Atl-7; lanes 17 and 18: Atl-8. The Atl number suffixes are indicated in Table 1 .

    Article Snippet: In addition, E. coli strain BL21 (EMD Millipore) was used for all protein expression studies.

    Techniques: Staining, Over Expression, Recombinant, SDS Page, Expressing

    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Binding Assay, Recombinant, Produced, Mutagenesis

    Identification of the luxS mutant BL21∆ luxS. a Schematic chart of strategy for producing the BL21 luxS deletion mutant. The luxS was deleted by replacing the partial gene sequence of luxS with kanamycin resistance cassette at Sal I cleavage sites. The primers used for the confirmation of the luxS deletion are also indicated. b Identification of the luxS mutant BL21∆ luxS. Lane M: DL 2000 DNA marker (D501A; Takara); lane 1: the wild-type strain BL21 showed a 2519-bp PCR product using primers LuxS-OutF/LuxS-OutR; lane 2: the mutant BL21∆ luxS with cure of the kanamycin resistance cassette showed a 2209-bp PCR product using primers LuxS-OutF/LuxS-OutR; lane 3: negative control; lane 4: the wild-type strain DE17 showed a 307-bp PCR product using primers LuxS-inF/LuxS-inR; lane 5: the mutant DE17∆ pfs showed no PCR products using primers LuxS-inF/LuxS-inR; lane 6: negative control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Identification of the luxS mutant BL21∆ luxS. a Schematic chart of strategy for producing the BL21 luxS deletion mutant. The luxS was deleted by replacing the partial gene sequence of luxS with kanamycin resistance cassette at Sal I cleavage sites. The primers used for the confirmation of the luxS deletion are also indicated. b Identification of the luxS mutant BL21∆ luxS. Lane M: DL 2000 DNA marker (D501A; Takara); lane 1: the wild-type strain BL21 showed a 2519-bp PCR product using primers LuxS-OutF/LuxS-OutR; lane 2: the mutant BL21∆ luxS with cure of the kanamycin resistance cassette showed a 2209-bp PCR product using primers LuxS-OutF/LuxS-OutR; lane 3: negative control; lane 4: the wild-type strain DE17 showed a 307-bp PCR product using primers LuxS-inF/LuxS-inR; lane 5: the mutant DE17∆ pfs showed no PCR products using primers LuxS-inF/LuxS-inR; lane 6: negative control

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Mutagenesis, Sequencing, Marker, Polymerase Chain Reaction, Negative Control

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis, Positive Control, Negative Control

    Identification of the luxS mutant BL21∆ luxS. a Schematic chart of strategy for producing the BL21 luxS deletion mutant. The luxS was deleted by replacing the partial gene sequence of luxS with kanamycin resistance cassette at Sal I cleavage sites.

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Identification of the luxS mutant BL21∆ luxS. a Schematic chart of strategy for producing the BL21 luxS deletion mutant. The luxS was deleted by replacing the partial gene sequence of luxS with kanamycin resistance cassette at Sal I cleavage sites.

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Mutagenesis, Sequencing

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis