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Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli <t>BL21</t> Star <t>DE3</t> and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.
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Article Title: Complex cellular logic computation using ribocomputing devices

Journal: Nature

doi: 10.1038/nature23271

Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli BL21 Star DE3 and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.
Figure Legend Snippet: Systematic study of AND gate circuit overlap domain lengths and comparison of two-input AND ribocomputing devices in different strains a , An early two-input AND gate was constructed from a standard toehold switch by dividing the trigger evenly into two 15-nt domains A1 and A2. Overlap domains u and u* were designed to cause the two input RNAs to hybridize and form an active trigger. b , A domain u’ was used to vary the region complementary to u* and measure its effect on expression levels. ON/OFF GFP ratios (left axis) vary as a function of the u’ domain length. The onset of substantial GFP expression coincides with the melting temperature of u’-u* hybridization rising above 37°C (right axis). c-f , Comparison of two-input AND ribocomputing devices in RNase-deficient E. coli BL21 Star DE3 and non-RNase-deficient E. coli BL21 DE3. c-d , ON/OFF GFP on linear (c) and logarithmic (d) scales measured for the two-input AND gate from Figure 2e–h . e-f , ON/OFF GFP on linear (e) and logarithmic (f) scales measured for a second two-input AND gate with an identical design but different RNA sequences.

Techniques Used: Construct, Expressing, Hybridization

Comparison of six-input OR gate ribocomputing devices measured in RNase-deficient E. coli (BL21 Star DE3) and non-RNase-deficient E. coli (BL21 DE3, MG1655Pro) a-b , ON/OFF GFP ratios measured for the device using T7 RNA polymerase in BL21 Star DE3 and BL21 DE3 cells on linear (a) and logarithmic (b) scales. Gate and input RNAs were expressed using the T7 RNA polymerase and measured 4 hours after induction with IPTG. c-d , ON/OFF GFP ratios obtained from the OR gate using E. coli RNA polymerase in MG1655Pro cells on linear (c) and logarithmic (d) scales. Gate and input RNAs were expressed using the E. coli RNA polymerase and measured 4 hours after induction of the gate RNA with IPTG. Input and decoy RNAs were expressed using a constitutive PN25 promoter.
Figure Legend Snippet: Comparison of six-input OR gate ribocomputing devices measured in RNase-deficient E. coli (BL21 Star DE3) and non-RNase-deficient E. coli (BL21 DE3, MG1655Pro) a-b , ON/OFF GFP ratios measured for the device using T7 RNA polymerase in BL21 Star DE3 and BL21 DE3 cells on linear (a) and logarithmic (b) scales. Gate and input RNAs were expressed using the T7 RNA polymerase and measured 4 hours after induction with IPTG. c-d , ON/OFF GFP ratios obtained from the OR gate using E. coli RNA polymerase in MG1655Pro cells on linear (c) and logarithmic (d) scales. Gate and input RNAs were expressed using the E. coli RNA polymerase and measured 4 hours after induction of the gate RNA with IPTG. Input and decoy RNAs were expressed using a constitutive PN25 promoter.

Techniques Used:

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Construct:

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Incubation:

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Expressing:

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Transformation Assay:

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Flow Cytometry:

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Ligation:

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Protease Inhibitor:

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Cell Culture:

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DNA Sequencing:

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Sequencing:

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Recombinant:

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Mutagenesis:

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Isolation:

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Subcloning:

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Purification:

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Protein Purification:

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Polymerase Chain Reaction:

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Article Title: Simple and efficient expression of Agaricus meleagris pyranose dehydrogenase in Pichia pastoris
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Affinity Chromatography:

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SPR Assay:

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Plasmid Preparation:

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Article Title: RNA remodeling by bacterial global regulator CsrA promotes Rho-dependent transcription termination
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Article Snippet: The pdh1 gene coding for pyranose dehydrogenase from the basidiomycete A. meleagris (GenBank accession number AY753307) has been previously isolated and cloned into the pCR Blunt II TOPO vector (Kittl et al. ). .. Escherichia coli strain DH5α-T1 was used for subcloning and BL21 DE3 for expression (Invitrogen).

Article Title: High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets
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Article Title: Domain Dissection and Characterization of the Aminoglycoside Resistance Enzyme ANT(3?)-Ii/AAC(6?)-IId from Serratia marcescens
Article Snippet: Chemically competent E. coli TOP10 and BL21(DE3) were from Invitrogen. .. The pET22b and pET28a vectors were from Novagen and the Int-pET19b-pps vector [ ] was obtained from Dr. Tapan Biswas (University of Michigan).

Selection:

Article Title: Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing
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Knock-Out:

Article Title: Characterization of an α-l-fucosidase from the periodontal pathogen Tannerella forsythia
Article Snippet: T. forsythia wild-type (WT) strain ATCC 43037 (American Type Culture Collection) and the knockout mutant Δ Tffuc1 were grown anaerobically at 37°C for 4–7 d in brain heart infusion (BHI) broth or 0.8% (w/v) BHI agar, supplemented with N ‑acetylmuramic acid (NAM), horse serum and gentamycin as described previously. .. Escherichia coli DH5α and BL21 (DE3) (Invitrogen) were cultivated in selective Luria Bertani (LB) medium (agar and broth) supplemented with 100 μg/ml ampicillin (Amp).

Concentration Assay:

Article Title: Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing
Article Snippet: The serC gene was disrupted in BL21 (DE3) (Invitrogen) to give BL21 (DE3) Δ serC by replacing the ORF of serC with a DNA fragment containing a chloramphenicol resistance gene (CmR) and sacB gene . .. The selection cassette was removed by recombination and selection on LB agar plates supplemented with sucrose to a concentration of 7.5% (w/v).

Positron Emission Tomography:

Article Title: High-throughput screening of microchip-synthesized genes in programmable double-emulsion droplets
Article Snippet: – To prepare the pET expression plasmid, synthetic gfp gene was inserted into pET-28a(+) (Novagen Inc., Madison, WI, USA) vector containing a lacI gene, a T7 promoter, a lac operator, an ampicillin resistance gene. .. Cloning product was transformed into BL21(DE3) chemically competent E. coli cells (Invitrogen) according to the manufacturer’s instruction.

Hood:

Article Title: Development of a serological assay to predict antibody bactericidal activity against non-typeable Haemophilus influenzae
Article Snippet: Bacterial strains and growth conditions NTHi strain 176 (kindly provided by Richard Moxon and Derek Hood, Oxford University, UK), isolated from a child with otitis media, was used for this study. .. Escherichia coli strains DH5α, HK100 and BL21 (DE3) (Invitrogen) were used for cloning and expression of NTHi antigens.

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  • 99
    Thermo Fisher e coli bl21
    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli <t>BL21</t> (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher plasmid interference assay bl21 ai
    Transcription-dependent loss of the target plasmid. <t>BL21-AI</t> strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.
    Plasmid Interference Assay Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher e coli bl21 cells
    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced <t>BL21</t> (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.
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    Thermo Fisher sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
    <t>SDS-PAGE</t> ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).
    Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Sds Page, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 497 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Journal: Molecular Cancer

    Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

    doi: 10.1186/s12943-017-0724-6

    Figure Lengend Snippet: KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Article Snippet: Briefly, pGEX-4T-1-KHSRP-WT was co-expressed with or without pE1E2SUMO1 plasmid in E.coli BL21 (DE3) respectively, and then lysed by using B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA) and incubated wi th Glutathione sepharose 4B (GE healthcare) at 4 °C overnight.

    Techniques: Modification, In Vitro, Transfection, Western Blot, Plasmid Preparation, Transformation Assay, Purification, Stripping Membranes, Mutagenesis, Construct

    Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

    Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Transformation Assay

    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Journal: Oncotarget

    Article Title: Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae

    doi: 10.18632/oncotarget.16475

    Figure Lengend Snippet: Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Article Snippet: The plasmids were then transformed into E. coli BL21 cells and induced by adding 1.0 mM IPTG at 37°C for 4 h. The cells were then centrifuged at 8000 × g for 10 min at 4°C, suspended in sterile phosphate-buffered saline (PBS), sonicated with a Sonic Dismembrator (model 500; Thermo Fisher Scientific, Waltham, MA, USA), and examined by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Techniques: Recombinant, Expressing, Purification, Marker, Polymerase Chain Reaction

    SDS-PAGE ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).

    Journal: Drug Target Insights

    Article Title: Molecular and Kinetic Characterization of Babesia microti Gray Strain Lactate Dehydrogenase as a Potential Drug Target

    doi: 10.4137/DTI.S16504

    Figure Lengend Snippet: SDS-PAGE ( A ) showing non-induced control (lane 1), expressed protein in pellet (lane 2), and supernatant/soluble fraction (lanes 3); SDS-PAGE ( B ) for purified GST-BmLDH protein (lanes 4) and PreScission ™ Protease cleaved BmLDH (lane 5).

    Article Snippet: The purity and concentration of the purified protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Lowry protein assay kit (Thermo Scientific, USA).

    Techniques: SDS Page, Purification