k lactis gg799  (New England Biolabs)


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    K lactis GG799 Competent Cells
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    K lactis GG799 Competent Cells 5 rxns
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    c1001s
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    Yeast Strains
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    New England Biolabs k lactis gg799
    K lactis GG799 Competent Cells
    K lactis GG799 Competent Cells 5 rxns
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    Average 95 stars, based on 12 article reviews
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    Images

    1) Product Images from "Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis"

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    Journal: Brazilian Journal of Microbiology

    doi: 10.1016/j.bjm.2017.10.001

    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    Figure Legend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Techniques Used: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    2) Product Images from "A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis"

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

    Journal: Fems Yeast Research

    doi: 10.1111/j.1567-1364.2010.00703.x

    Secretion of human interferon Hy3 by protease-deficient strains. (a) Western blot analysis of recombinant Hy3 present in SCM derived from strain GG799 (wt) (± an expression vector) and each protease mutant. (b, c) Analysis of Hy3 secreted by the Kluyveromyces lactis Δ yps1 mutant grown to high cell density in a bioreactor. Hy3 secreted after 22.5 and 40.5 h of fermentation was visualized by SDS-PAGE separation of SCM and either Coomassie blue staining (b) or Western blotting (c). SCM from GG799 cells not expressing Hy3 was analyzed as a negative control. In all panels, arrows indicate intact Hy3 protein and M represents a molecular weight marker. In (a) and (c), the asterisk denotes a proteolytic product of Hy3.
    Figure Legend Snippet: Secretion of human interferon Hy3 by protease-deficient strains. (a) Western blot analysis of recombinant Hy3 present in SCM derived from strain GG799 (wt) (± an expression vector) and each protease mutant. (b, c) Analysis of Hy3 secreted by the Kluyveromyces lactis Δ yps1 mutant grown to high cell density in a bioreactor. Hy3 secreted after 22.5 and 40.5 h of fermentation was visualized by SDS-PAGE separation of SCM and either Coomassie blue staining (b) or Western blotting (c). SCM from GG799 cells not expressing Hy3 was analyzed as a negative control. In all panels, arrows indicate intact Hy3 protein and M represents a molecular weight marker. In (a) and (c), the asterisk denotes a proteolytic product of Hy3.

    Techniques Used: Western Blot, Recombinant, Derivative Assay, Expressing, Plasmid Preparation, Mutagenesis, SDS Page, Staining, Negative Control, Molecular Weight, Marker

    3) Product Images from "A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis"

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00542-19

    Gluc expression and glucose consumption during culturing of strains containing hybrid promoter-Gluc constructs. K. lactis GG799 cells harboring P GAP1 -Gluc, P ICL1 -Gluc, P 1000 -Gluc, P 350 -Gluc, and P 125 -Gluc expression constructs were grown in YDFM containing glucose in bioreactors. Shown are (A) luciferase activity (RLU/100,000) and cell growth (OD 600 ) relative to hours of culturing, and (B) glucose consumption (%) and cell growth (OD 600 ) relative to hours of culturing. P 1000 -Gluc showed constitutive Gluc expression higher than that of P ICL1 -Gluc or P GAP1 -Gluc. P 350 -Gluc showed Gluc expression only after glucose had been consumed from the culture medium. No Gluc expression was observed with P 125 -Gluc. ND represents the glucose detection limit (
    Figure Legend Snippet: Gluc expression and glucose consumption during culturing of strains containing hybrid promoter-Gluc constructs. K. lactis GG799 cells harboring P GAP1 -Gluc, P ICL1 -Gluc, P 1000 -Gluc, P 350 -Gluc, and P 125 -Gluc expression constructs were grown in YDFM containing glucose in bioreactors. Shown are (A) luciferase activity (RLU/100,000) and cell growth (OD 600 ) relative to hours of culturing, and (B) glucose consumption (%) and cell growth (OD 600 ) relative to hours of culturing. P 1000 -Gluc showed constitutive Gluc expression higher than that of P ICL1 -Gluc or P GAP1 -Gluc. P 350 -Gluc showed Gluc expression only after glucose had been consumed from the culture medium. No Gluc expression was observed with P 125 -Gluc. ND represents the glucose detection limit (

    Techniques Used: Expressing, Construct, Luciferase, Activity Assay

    Gaussia luciferase (A) or α- l -fucosidase (B) expression in K. lactis in response to different promoters and carbon sources. For each strain, the average protein production from five individual cultures, each inoculated with a single transformant, is shown. Cells were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C. Ethanol was added to “glucose + ethanol” cultures after 24 h. Protein production at 48 h was determined by normalizing the activity of the reporter protein (RLU for luciferase or OD 405 for α- l -fucosidase) to the corresponding cell density (OD 600 ) of each culture. Cultures of the wild-type K. lactis GG799 strain were used as negative controls. Error bars represent the standard deviation of the mean of five different cultures.
    Figure Legend Snippet: Gaussia luciferase (A) or α- l -fucosidase (B) expression in K. lactis in response to different promoters and carbon sources. For each strain, the average protein production from five individual cultures, each inoculated with a single transformant, is shown. Cells were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C. Ethanol was added to “glucose + ethanol” cultures after 24 h. Protein production at 48 h was determined by normalizing the activity of the reporter protein (RLU for luciferase or OD 405 for α- l -fucosidase) to the corresponding cell density (OD 600 ) of each culture. Cultures of the wild-type K. lactis GG799 strain were used as negative controls. Error bars represent the standard deviation of the mean of five different cultures.

    Techniques Used: Luciferase, Expressing, Activity Assay, Standard Deviation

    Gaussia luciferase expression using the P ICL1 -P GAP1 fusion promoters in response to different carbon sources. (A) Diagrams of promoters P ICL1 , P GAP1 , P 1000 , and P 350 . The region between nucleotides −690 and −811 upstream from the ICL1 translation start site contains two carbon source-responsive elements (CSRE; shown in dark gray). A series of promoters was created by fusing the regulatory portion of P ICL1 to progressively shorter regions of the P GAP1 promoter relative to the GAP1 translation start site (i.e., P 1000 , P 500 , P 450 , P 400 , P 350 , P 220 , P 125 , and P 74 ). Shown in panel A, P 350 contains 350 nucleotides immediately upstream of the GAP1 translation start site fused to the P ICL1 regulatory fragment. (B) Average protein production from 10 individual strains harboring each hybrid promoter fused to DNA encoding Gaussia luciferase (Gluc). Control strains where four biological replicates were analyzed are indicated by the # symbol. All cultures were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C, after which Gluc activity (RLU) was normalized to the cell density (OD 600 ) for each culture. Cultures of wild-type K. lactis GG799 cells were used as negative controls. Error bars represent the standard deviation from the mean.
    Figure Legend Snippet: Gaussia luciferase expression using the P ICL1 -P GAP1 fusion promoters in response to different carbon sources. (A) Diagrams of promoters P ICL1 , P GAP1 , P 1000 , and P 350 . The region between nucleotides −690 and −811 upstream from the ICL1 translation start site contains two carbon source-responsive elements (CSRE; shown in dark gray). A series of promoters was created by fusing the regulatory portion of P ICL1 to progressively shorter regions of the P GAP1 promoter relative to the GAP1 translation start site (i.e., P 1000 , P 500 , P 450 , P 400 , P 350 , P 220 , P 125 , and P 74 ). Shown in panel A, P 350 contains 350 nucleotides immediately upstream of the GAP1 translation start site fused to the P ICL1 regulatory fragment. (B) Average protein production from 10 individual strains harboring each hybrid promoter fused to DNA encoding Gaussia luciferase (Gluc). Control strains where four biological replicates were analyzed are indicated by the # symbol. All cultures were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C, after which Gluc activity (RLU) was normalized to the cell density (OD 600 ) for each culture. Cultures of wild-type K. lactis GG799 cells were used as negative controls. Error bars represent the standard deviation from the mean.

    Techniques Used: Luciferase, Expressing, Activity Assay, Standard Deviation

    4) Product Images from "Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis"

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    Journal: Brazilian Journal of Microbiology

    doi: 10.1016/j.bjm.2017.10.001

    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    Figure Legend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Techniques Used: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    5) Product Images from "Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis"

    Article Title: Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

    Journal:

    doi: 10.1128/AEM.71.11.7092-7098.2005

    Activity of secreted enterokinase in the spent culture medium of K. lactis cells containing integrated pKLAC1-EK L . Seven K. lactis strains harboring pKLAC1-EK L and wild-type GG799 cells were grown in YPGal medium for 48 h. Cleared spent culture
    Figure Legend Snippet: Activity of secreted enterokinase in the spent culture medium of K. lactis cells containing integrated pKLAC1-EK L . Seven K. lactis strains harboring pKLAC1-EK L and wild-type GG799 cells were grown in YPGal medium for 48 h. Cleared spent culture

    Techniques Used: Activity Assay

    6) Product Images from "Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis"

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    Journal: Brazilian Journal of Microbiology

    doi: 10.1016/j.bjm.2017.10.001

    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    Figure Legend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Techniques Used: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    7) Product Images from "Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis"

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    Journal: Brazilian Journal of Microbiology

    doi: 10.1016/j.bjm.2017.10.001

    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    Figure Legend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Techniques Used: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    8) Product Images from "Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis"

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.6.2862-2869.2005

    (A) K. lactis Δ cts1 cells do not secrete KlCts1p. Proteins in spent culture medium from wild-type GG799 (WT) and Δ cts1 K. lactis cells were incubated with chitin beads. Bound proteins were eluted by boiling and were detected by α-ChBD
    Figure Legend Snippet: (A) K. lactis Δ cts1 cells do not secrete KlCts1p. Proteins in spent culture medium from wild-type GG799 (WT) and Δ cts1 K. lactis cells were incubated with chitin beads. Bound proteins were eluted by boiling and were detected by α-ChBD

    Techniques Used: Incubation

    Identification of a secreted 85-kDa K. lactis chitin-binding protein. Secreted proteins in 10 μl of K. lactis GG799 spent culture medium (after 96 h of growth) were separated by SDS-PAGE and screened for the presence of a chitin-binding domain
    Figure Legend Snippet: Identification of a secreted 85-kDa K. lactis chitin-binding protein. Secreted proteins in 10 μl of K. lactis GG799 spent culture medium (after 96 h of growth) were separated by SDS-PAGE and screened for the presence of a chitin-binding domain

    Techniques Used: Binding Assay, SDS Page

    Enzymatic properties of secreted KlCts1p. (A) Substrate preferences of KlCts1p and ScCts1p. Spent culture media from cultures of K. lactis GG799 and S. cerevisiae BY4741 cells were incubated with 50 μM 4MU-GlcNAc 3 (Tri) (black bars) and with 50
    Figure Legend Snippet: Enzymatic properties of secreted KlCts1p. (A) Substrate preferences of KlCts1p and ScCts1p. Spent culture media from cultures of K. lactis GG799 and S. cerevisiae BY4741 cells were incubated with 50 μM 4MU-GlcNAc 3 (Tri) (black bars) and with 50

    Techniques Used: Incubation

    9) Product Images from "Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis"

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    Journal: Brazilian Journal of Microbiology

    doi: 10.1016/j.bjm.2017.10.001

    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    Figure Legend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Techniques Used: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    Related Articles

    Clone Assay:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Escherichia coli XL1-Blue [recA1 endA1 gyrA96 thi -1 hsdR17 supE44 relA1 lac [F’proAB lacIqZDM15 Tn10 (Tetr)] ] (Stratagene Cloning Systems) was used as host for standard recombinant DNA techniques [ ].

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Mutation, expression, purification and crystallization The coding sequence of human serum albumin (corresponding to residues 19-609 of the prepro-albumin sequence; NP_000468) was amplified and cloned into pKLAC2 plasmid using Xho /NotI restriction sites downstream and in frame with the α-mating factor secretion signal sequence. .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning. .. The pGEM-T Easy (Promega, Madison, WI, USA) and pKLAC2 vectors were used for gene cloning in E. coli and for gene expression in K. lactis , respectively.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning. .. The pGEM-T Easy (Promega, Madison, WI, USA) and pKLAC2 vectors were used for gene cloning in E. coli and for gene expression in K. lactis , respectively.

    Article Title: Immunological Comparison of Native and Recombinant Hen’s Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis
    Article Snippet: Strains, Vectors, and Growth Conditions Escherichia coli (E. coli ) strain NEB® 5-alpha F’ I q (New England Biolabs Inc., Ipswich, MA, USA) was used as the cloning host and was grown in Luria-Bertani (LB) medium supplemented with 50 mg/mL ampicillin at 37 °C. .. K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA.

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: .. Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis .

    Centrifugation:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water. .. Approximately 50 μl of protein-bound chitin beads was removed and boiled in SDS sample buffer (New England Biolabs) for 2 min to elute bound proteins, after which the mixture was microcentrifuged for 2 min to remove the chitin beads.

    Amplification:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Phusion High-Fidelity DNA Polymerase (for gene amplification), DreamTaq polymerase (for colony PCR analysis), and the DNA purification kits GeneJEt Gel Extraction Kit and GeneJET Plasmid Miniprep Kit, were used following the specifications of the supplier (Fisher Scientific).

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Mutation, expression, purification and crystallization The coding sequence of human serum albumin (corresponding to residues 19-609 of the prepro-albumin sequence; NP_000468) was amplified and cloned into pKLAC2 plasmid using Xho /NotI restriction sites downstream and in frame with the α-mating factor secretion signal sequence. .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: .. Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis .

    Construct:

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Oligonucleotide directed mutagenesis was used to prepare a construct encoding the mutated albumin (H67A) using the QuikChange Site-Directed Mutagenesis Kit (Agilent, Cheshire, UK). .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799 The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The expression vector pKLAC2- Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Incubation:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. Null mutants lacking the amdS gene were then isolated by three rounds of restreaking on YCB agar supplemented with 10 mM fluoroacetamide (Sigma-Aldrich) and 0.1% (w/v) ammonium sulfate, and incubation for 2–3 days at 30 °C.

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water. .. Approximately 50 μl of protein-bound chitin beads was removed and boiled in SDS sample buffer (New England Biolabs) for 2 min to elute bound proteins, after which the mixture was microcentrifuged for 2 min to remove the chitin beads.

    Activity Assay:

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Transformants were selected on YCB medium (1.17% yeast carbon base, 0.03 M sodium phosphate buffer, pH 7, New England Biolabs) with 5 mM acetamide and were grown on YPD plates containing 1% RBB-xylan for activity screening.

    Expressing:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: .. Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Escherichia coli XL1-Blue [recA1 endA1 gyrA96 thi -1 hsdR17 supE44 relA1 lac [F’proAB lacIqZDM15 Tn10 (Tetr)] ] (Stratagene Cloning Systems) was used as host for standard recombinant DNA techniques [ ].

    Article Title: Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis
    Article Snippet: .. Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier. .. Colonies of cells transformed with pGBN1, pGBN1PGK1 , pGBN1Hyb , pGBN1PBI , or pGBN1PBII-PBIII vectors were selected by growth on YPD agar plates containing 200 μg G418 (Sigma, St. Louis, MO) ml−1 for 2 to 3 days at 30°C.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The transformation of the expression cassette into K. lactis GG799 was performed using lithium acetate with the heat shock method. .. To integrate the cloned endoglucanase gene into the LAC4 locus of the K. lactis chromosome, the expression cassette pKLAC2-Cel7 was linearized with Bst XI before transformation.

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Paragraph title: Mutation, expression, purification and crystallization ... The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning. .. The pGEM-T Easy (Promega, Madison, WI, USA) and pKLAC2 vectors were used for gene cloning in E. coli and for gene expression in K. lactis , respectively.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799 The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The expression vector pKLAC2- Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning. .. The pGEM-T Easy (Promega, Madison, WI, USA) and pKLAC2 vectors were used for gene cloning in E. coli and for gene expression in K. lactis , respectively.

    Article Title: Immunological Comparison of Native and Recombinant Hen’s Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis
    Article Snippet: K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA. .. The K. lactis integrative expression vector pKLAC2 (New England Biolabs Inc., Ipswich, MA, USA) contains the Aspergillus nidulans acetamidase gene (amdS ), which allows growth on nitrogen-free minimal medium containing acetamide.

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: .. Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis .

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: .. For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs). .. Transformants were selected by growth on nitrogen-free yeast carbon base (YCB) agar medium (New England Biolabs) supplemented with 5 mM acetamide for 3 to 4 days at 30°C.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The transformation of the expression cassette into K. lactis GG799 was performed using lithium acetate with the heat shock method. .. To integrate the cloned endoglucanase gene into the LAC4 locus of the K. lactis chromosome, the expression cassette pKLAC2- Cel7 was linearized with Bst XI before transformation.

    Western Blot:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: Western blotting was used to detect secreted K. lactis proteins that cross-reacted with a polyclonal anti-chitin-binding domain antibody (α-ChBD) raised against the chitin-binding domain derived from Bacillus circulans chitinase A1 (New England Biolabs, Beverly, MA). .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water.

    Transformation Assay:

    Article Title: Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis
    Article Snippet: .. Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier. .. Colonies of cells transformed with pGBN1, pGBN1PGK1 , pGBN1Hyb , pGBN1PBI , or pGBN1PBII-PBIII vectors were selected by growth on YPD agar plates containing 200 μg G418 (Sigma, St. Louis, MO) ml−1 for 2 to 3 days at 30°C.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The transformation of the expression cassette into K. lactis GG799 was performed using lithium acetate with the heat shock method. .. To integrate the cloned endoglucanase gene into the LAC4 locus of the K. lactis chromosome, the expression cassette pKLAC2-Cel7 was linearized with Bst XI before transformation.

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection. .. Genomic DNA was extracted from resultant clones using the Wizard Yeast Genomic DNA Purification Kit (Promega, Southampton, UK) and correct insertion of the expression cassette into the K. lactis genome was verified by PCR, using primers flanking the insertion site.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799 The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The expression vector pKLAC2- Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Immunological Comparison of Native and Recombinant Hen’s Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis
    Article Snippet: K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA. .. Selection of K. lactis cells transformed with pKLAC2 vectors was performed by growth on yeast carbon base (YCB) agar medium with 5 mM acetamide at 30 °C.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The transformation of the expression cassette into K. lactis GG799 was performed using lithium acetate with the heat shock method. .. To integrate the cloned endoglucanase gene into the LAC4 locus of the K. lactis chromosome, the expression cassette pKLAC2- Cel7 was linearized with Bst XI before transformation.

    Crystallization Assay:

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Paragraph title: Mutation, expression, purification and crystallization ... The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Derivative Assay:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: Western blotting was used to detect secreted K. lactis proteins that cross-reacted with a polyclonal anti-chitin-binding domain antibody (α-ChBD) raised against the chitin-binding domain derived from Bacillus circulans chitinase A1 (New England Biolabs, Beverly, MA). .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water.

    Northern Blot:

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Cell Culture:

    Article Title: Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis
    Article Snippet: Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier. .. K. lactis strains expressing heterologous genes were cultured in YP medium containing 2% galactose (YPGal) at 30°C for 48 to 96 h.

    DNA Sequencing:

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs). .. Strains were verified using PCR and genomic DNA sequencing.

    Polymerase Chain Reaction:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. Successful disruption of a target chromosomal locus was assessed by whole-cell PCR (see previous section).

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Phusion High-Fidelity DNA Polymerase (for gene amplification), DreamTaq polymerase (for colony PCR analysis), and the DNA purification kits GeneJEt Gel Extraction Kit and GeneJET Plasmid Miniprep Kit, were used following the specifications of the supplier (Fisher Scientific).

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection. .. Genomic DNA was extracted from resultant clones using the Wizard Yeast Genomic DNA Purification Kit (Promega, Southampton, UK) and correct insertion of the expression cassette into the K. lactis genome was verified by PCR, using primers flanking the insertion site.

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs). .. Strains were verified using PCR and genomic DNA sequencing.

    Recombinant:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: .. Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Escherichia coli XL1-Blue [recA1 endA1 gyrA96 thi -1 hsdR17 supE44 relA1 lac [F’proAB lacIqZDM15 Tn10 (Tetr)] ] (Stratagene Cloning Systems) was used as host for standard recombinant DNA techniques [ ].

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: .. Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis .

    Nucleic Acid Electrophoresis:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: Spent culture medium was isolated from K. lactis GG799 cultures following 48 to 96 h of growth by centrifugation at 4,000 × g for 10 min. Proteins in the spent medium were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on a 4 to 20% Tris-glycine polyacrylamide gel (Daiichi Pure Chemicals Co., Tokyo, Japan) and were transferred to a Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH). .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water.

    Mutagenesis:

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Paragraph title: Mutation, expression, purification and crystallization ... The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Isolation:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. Null mutants lacking the amdS gene were then isolated by three rounds of restreaking on YCB agar supplemented with 10 mM fluoroacetamide (Sigma-Aldrich) and 0.1% (w/v) ammonium sulfate, and incubation for 2–3 days at 30 °C.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: It was isolated from a soil sample collected in the northern part of Thailand, and was identified by morphological analysis and DNA sequencing of the internal transcribed spacer (ITS) and large subunit (LSU) of rDNA region. .. K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: Paragraph title: Detection and isolation of secreted K. lactis chitin-binding proteins. ... To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water.

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs). .. Genomic DNA was isolated using a lithium acetate-sodium dodecyl sulfate (LiOAc-SDS) recovery method ( ).

    Purification:

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Paragraph title: Mutation, expression, purification and crystallization ... The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: .. For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs). .. Transformants were selected by growth on nitrogen-free yeast carbon base (YCB) agar medium (New England Biolabs) supplemented with 5 mM acetamide for 3 to 4 days at 30°C.

    Sequencing:

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Mutation, expression, purification and crystallization The coding sequence of human serum albumin (corresponding to residues 19-609 of the prepro-albumin sequence; NP_000468) was amplified and cloned into pKLAC2 plasmid using Xho /NotI restriction sites downstream and in frame with the α-mating factor secretion signal sequence. .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water. .. The supernatant, containing eluted proteins, was separated by SDS-PAGE and subjected either to Western analysis with α-ChBD polyclonal antibodies or to amino-terminal protein sequencing.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: Spent culture medium was isolated from K. lactis GG799 cultures following 48 to 96 h of growth by centrifugation at 4,000 × g for 10 min. Proteins in the spent medium were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) on a 4 to 20% Tris-glycine polyacrylamide gel (Daiichi Pure Chemicals Co., Tokyo, Japan) and were transferred to a Protran nitrocellulose membrane (Schleicher & Schuell, Keene, NH). .. To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water.

    Polymerase Cycling Assembly:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: .. Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. Successful disruption of a target chromosomal locus was assessed by whole-cell PCR (see previous section).

    SDS Page:

    Article Title: Characterization of a Nucleus-Encoded Chitinase from the Yeast Kluyveromyces lactis
    Article Snippet: To isolate secreted proteins that bind chitin, K. lactis GG799 cells were grown in 20 ml YPD medium for 96 h. Cells were removed from the culture by centrifugation, and the spent medium was transferred to a fresh tube containing 1 ml of water-washed chitin beads (New England Biolabs) and incubated at room temperature with gentle rotation for 1 h. The chitin beads were harvested by centrifugation, washed with 10 ml of water, and resuspended in 1 ml of water. .. The supernatant, containing eluted proteins, was separated by SDS-PAGE and subjected either to Western analysis with α-ChBD polyclonal antibodies or to amino-terminal protein sequencing.

    Plasmid Preparation:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. The plasmid YEpMEL1 His [ampr ori 2μ MEL1 His TRP1 ] [ ], was used as template to amplify the gene fusion.

    Article Title: Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis
    Article Snippet: Prior to the transformation of GG799 cells, 1 μg of pGBN1- or pKLAC1-based expression vector containing a gene of interest was linearized by SacII digestion. .. Linearized expression vectors were used for the integrative transformation of chemically competent K. lactis GG799 cells (New England Biolabs, Ipswich, MA) as directed by the supplier.

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: Mutation, expression, purification and crystallization The coding sequence of human serum albumin (corresponding to residues 19-609 of the prepro-albumin sequence; NP_000468) was amplified and cloned into pKLAC2 plasmid using Xho /NotI restriction sites downstream and in frame with the α-mating factor secretion signal sequence. .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: Paragraph title: Strains, plasmid vectors and culture conditions ... K. lactis GG799 (New England Biolabs, US) was used as a host system for protein expression, while Escherichia coli DH5α was used as a host system for gene cloning.

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799 The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis
    Article Snippet: .. The expression vector pKLAC2- Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section. ..

    Article Title: Immunological Comparison of Native and Recombinant Hen’s Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis
    Article Snippet: K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA. .. The K. lactis integrative expression vector pKLAC2 (New England Biolabs Inc., Ipswich, MA, USA) contains the Aspergillus nidulans acetamidase gene (amdS ), which allows growth on nitrogen-free minimal medium containing acetamide.

    Article Title: Developing a xylanase XYNZG from Plectosphaerella cucumerina for baking by heterologously expressed in Kluyveromyces lactis
    Article Snippet: .. Strains, plasmids and media The strain K. lactis GG799 (New England Biolabs, USA) was used as the host for the expression of recombinant protein and E. coli DH5α was used as the host for cloning and plasmid amplification. .. Plasmid pKLAC2 (New England Biolabs, USA) was employed as the expression vector of K. lactis .

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: Paragraph title: Plasmid and strain construction. ... For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs).

    Selection:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: .. Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. Successful disruption of a target chromosomal locus was assessed by whole-cell PCR (see previous section).

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: .. The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection. .. Genomic DNA was extracted from resultant clones using the Wizard Yeast Genomic DNA Purification Kit (Promega, Southampton, UK) and correct insertion of the expression cassette into the K. lactis genome was verified by PCR, using primers flanking the insertion site.

    Article Title: Immunological Comparison of Native and Recombinant Hen’s Egg Yolk Allergen, Chicken Serum Albumin (Gal d 5), Produced in Kluveromyces lactis
    Article Snippet: K. lactis strain GG799 (New England Biolabs Inc., Ipswich, MA, USA) was used as the host strain for the secretion of rCSA. .. Selection of K. lactis cells transformed with pKLAC2 vectors was performed by growth on yeast carbon base (YCB) agar medium with 5 mM acetamide at 30 °C.

    In Vitro:

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis
    Article Snippet: A 2-μl aliquot of the in vitro assembly reaction mixture was used to transform 5-alpha competent E. coli cells (New England Biolabs). .. For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs).

    DNA Purification:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Phusion High-Fidelity DNA Polymerase (for gene amplification), DreamTaq polymerase (for colony PCR analysis), and the DNA purification kits GeneJEt Gel Extraction Kit and GeneJET Plasmid Miniprep Kit, were used following the specifications of the supplier (Fisher Scientific).

    Article Title: Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins zinc transport is controlled by distinct interdomain sites on mammalian albumins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc02267gClick here for additional data file.
    Article Snippet: The resultant plasmids were linearized using BstXI enzyme and transformed into Kluyveromyces lactis GG799 competent cells (New England Biolabs, Hitchin, UK) in accordance with the manufacturer's instructions and grown under acetamide selection. .. Genomic DNA was extracted from resultant clones using the Wizard Yeast Genomic DNA Purification Kit (Promega, Southampton, UK) and correct insertion of the expression cassette into the K. lactis genome was verified by PCR, using primers flanking the insertion site.

    Marker:

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis
    Article Snippet: Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C. .. To recycle the amdS marker, a strain harboring an amdS + disrupted target allele was grown in the absence of selection on YPD agar to permit recombination between the directly repeating UTR regions that flank the amdS gene ( ).

    Gel Extraction:

    Article Title: Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase
    Article Snippet: Strains and other materials for recombinant DNA techniques The K. lactis strains GG799 {Matα [pGKl1 +]} (New England, Biolabs) and NRRL-Y1140 {Matα} [ ] were chosen for the integration and expression of the gene encoding the fusion protein of interest (MEL1 His). .. Phusion High-Fidelity DNA Polymerase (for gene amplification), DreamTaq polymerase (for colony PCR analysis), and the DNA purification kits GeneJEt Gel Extraction Kit and GeneJET Plasmid Miniprep Kit, were used following the specifications of the supplier (Fisher Scientific).

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    New England Biolabs k lactis gg799
    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. <t>lactis</t> <t>GG799;</t> 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.
    K Lactis Gg799, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Journal: Brazilian Journal of Microbiology

    Article Title: Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

    doi: 10.1016/j.bjm.2017.10.001

    Figure Lengend Snippet: SDS-PAGE and zymogram analysis of the endoglucanase (A) and the purified recombinant endoglucanase (B) from A. fumigatus DBiNU-1. The protein samples (30 μg) were loaded on the gel and subjected to electrophoresis. (A) M, protein marker; 1, the cell-free supernatant from K. lactis GG799; 2, the cell-free supernatant from K. lactis harboring pKLAC2- Cel7 ; 3, zymogram analysis of the endoglucanase after Congo red staining. (B) M, protein marker; 1, crude recombinant endoglucanase; 2, purified recombinant endoglucanase.

    Article Snippet: Expression of the endoglucanase gene from A. fumigatus DBiNU-1 in K. lactis GG799 The expression vector pKLAC2-Cel7 was constructed and transformed into K. lactis GG799, and the transformants were selected using YCB agar medium, as described in the Materials and methods section.

    Techniques: SDS Page, Purification, Recombinant, Electrophoresis, Marker, Staining

    Secretion of human interferon Hy3 by protease-deficient strains. (a) Western blot analysis of recombinant Hy3 present in SCM derived from strain GG799 (wt) (± an expression vector) and each protease mutant. (b, c) Analysis of Hy3 secreted by the Kluyveromyces lactis Δ yps1 mutant grown to high cell density in a bioreactor. Hy3 secreted after 22.5 and 40.5 h of fermentation was visualized by SDS-PAGE separation of SCM and either Coomassie blue staining (b) or Western blotting (c). SCM from GG799 cells not expressing Hy3 was analyzed as a negative control. In all panels, arrows indicate intact Hy3 protein and M represents a molecular weight marker. In (a) and (c), the asterisk denotes a proteolytic product of Hy3.

    Journal: Fems Yeast Research

    Article Title: A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis

    doi: 10.1111/j.1567-1364.2010.00703.x

    Figure Lengend Snippet: Secretion of human interferon Hy3 by protease-deficient strains. (a) Western blot analysis of recombinant Hy3 present in SCM derived from strain GG799 (wt) (± an expression vector) and each protease mutant. (b, c) Analysis of Hy3 secreted by the Kluyveromyces lactis Δ yps1 mutant grown to high cell density in a bioreactor. Hy3 secreted after 22.5 and 40.5 h of fermentation was visualized by SDS-PAGE separation of SCM and either Coomassie blue staining (b) or Western blotting (c). SCM from GG799 cells not expressing Hy3 was analyzed as a negative control. In all panels, arrows indicate intact Hy3 protein and M represents a molecular weight marker. In (a) and (c), the asterisk denotes a proteolytic product of Hy3.

    Article Snippet: Construction of protease-deficient strains After its assembly by PCR, 2 μg of a gene disruption DNA fragment was introduced into K. lactis GG799 competent cells as described by the manufacturer (New England Biolabs) followed by selection of transformants by growth on YCB agar medium supplemented with 5 mM acetamide for no more than 3 days at 30 °C.

    Techniques: Western Blot, Recombinant, Derivative Assay, Expressing, Plasmid Preparation, Mutagenesis, SDS Page, Staining, Negative Control, Molecular Weight, Marker

    Gluc expression and glucose consumption during culturing of strains containing hybrid promoter-Gluc constructs. K. lactis GG799 cells harboring P GAP1 -Gluc, P ICL1 -Gluc, P 1000 -Gluc, P 350 -Gluc, and P 125 -Gluc expression constructs were grown in YDFM containing glucose in bioreactors. Shown are (A) luciferase activity (RLU/100,000) and cell growth (OD 600 ) relative to hours of culturing, and (B) glucose consumption (%) and cell growth (OD 600 ) relative to hours of culturing. P 1000 -Gluc showed constitutive Gluc expression higher than that of P ICL1 -Gluc or P GAP1 -Gluc. P 350 -Gluc showed Gluc expression only after glucose had been consumed from the culture medium. No Gluc expression was observed with P 125 -Gluc. ND represents the glucose detection limit (

    Journal: Applied and Environmental Microbiology

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis

    doi: 10.1128/AEM.00542-19

    Figure Lengend Snippet: Gluc expression and glucose consumption during culturing of strains containing hybrid promoter-Gluc constructs. K. lactis GG799 cells harboring P GAP1 -Gluc, P ICL1 -Gluc, P 1000 -Gluc, P 350 -Gluc, and P 125 -Gluc expression constructs were grown in YDFM containing glucose in bioreactors. Shown are (A) luciferase activity (RLU/100,000) and cell growth (OD 600 ) relative to hours of culturing, and (B) glucose consumption (%) and cell growth (OD 600 ) relative to hours of culturing. P 1000 -Gluc showed constitutive Gluc expression higher than that of P ICL1 -Gluc or P GAP1 -Gluc. P 350 -Gluc showed Gluc expression only after glucose had been consumed from the culture medium. No Gluc expression was observed with P 125 -Gluc. ND represents the glucose detection limit (

    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs).

    Techniques: Expressing, Construct, Luciferase, Activity Assay

    Gaussia luciferase (A) or α- l -fucosidase (B) expression in K. lactis in response to different promoters and carbon sources. For each strain, the average protein production from five individual cultures, each inoculated with a single transformant, is shown. Cells were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C. Ethanol was added to “glucose + ethanol” cultures after 24 h. Protein production at 48 h was determined by normalizing the activity of the reporter protein (RLU for luciferase or OD 405 for α- l -fucosidase) to the corresponding cell density (OD 600 ) of each culture. Cultures of the wild-type K. lactis GG799 strain were used as negative controls. Error bars represent the standard deviation of the mean of five different cultures.

    Journal: Applied and Environmental Microbiology

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis

    doi: 10.1128/AEM.00542-19

    Figure Lengend Snippet: Gaussia luciferase (A) or α- l -fucosidase (B) expression in K. lactis in response to different promoters and carbon sources. For each strain, the average protein production from five individual cultures, each inoculated with a single transformant, is shown. Cells were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C. Ethanol was added to “glucose + ethanol” cultures after 24 h. Protein production at 48 h was determined by normalizing the activity of the reporter protein (RLU for luciferase or OD 405 for α- l -fucosidase) to the corresponding cell density (OD 600 ) of each culture. Cultures of the wild-type K. lactis GG799 strain were used as negative controls. Error bars represent the standard deviation of the mean of five different cultures.

    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs).

    Techniques: Luciferase, Expressing, Activity Assay, Standard Deviation

    Gaussia luciferase expression using the P ICL1 -P GAP1 fusion promoters in response to different carbon sources. (A) Diagrams of promoters P ICL1 , P GAP1 , P 1000 , and P 350 . The region between nucleotides −690 and −811 upstream from the ICL1 translation start site contains two carbon source-responsive elements (CSRE; shown in dark gray). A series of promoters was created by fusing the regulatory portion of P ICL1 to progressively shorter regions of the P GAP1 promoter relative to the GAP1 translation start site (i.e., P 1000 , P 500 , P 450 , P 400 , P 350 , P 220 , P 125 , and P 74 ). Shown in panel A, P 350 contains 350 nucleotides immediately upstream of the GAP1 translation start site fused to the P ICL1 regulatory fragment. (B) Average protein production from 10 individual strains harboring each hybrid promoter fused to DNA encoding Gaussia luciferase (Gluc). Control strains where four biological replicates were analyzed are indicated by the # symbol. All cultures were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C, after which Gluc activity (RLU) was normalized to the cell density (OD 600 ) for each culture. Cultures of wild-type K. lactis GG799 cells were used as negative controls. Error bars represent the standard deviation from the mean.

    Journal: Applied and Environmental Microbiology

    Article Title: A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression in Kluyveromyces lactis

    doi: 10.1128/AEM.00542-19

    Figure Lengend Snippet: Gaussia luciferase expression using the P ICL1 -P GAP1 fusion promoters in response to different carbon sources. (A) Diagrams of promoters P ICL1 , P GAP1 , P 1000 , and P 350 . The region between nucleotides −690 and −811 upstream from the ICL1 translation start site contains two carbon source-responsive elements (CSRE; shown in dark gray). A series of promoters was created by fusing the regulatory portion of P ICL1 to progressively shorter regions of the P GAP1 promoter relative to the GAP1 translation start site (i.e., P 1000 , P 500 , P 450 , P 400 , P 350 , P 220 , P 125 , and P 74 ). Shown in panel A, P 350 contains 350 nucleotides immediately upstream of the GAP1 translation start site fused to the P ICL1 regulatory fragment. (B) Average protein production from 10 individual strains harboring each hybrid promoter fused to DNA encoding Gaussia luciferase (Gluc). Control strains where four biological replicates were analyzed are indicated by the # symbol. All cultures were grown in deep-well plates containing YEP medium supplemented with different carbon sources for 48 h at 30°C, after which Gluc activity (RLU) was normalized to the cell density (OD 600 ) for each culture. Cultures of wild-type K. lactis GG799 cells were used as negative controls. Error bars represent the standard deviation from the mean.

    Article Snippet: For expression in yeast, a purified linearized expression cassette (0.1 μg) was introduced into K. lactis GG799 competent cells, as described by the manufacturer (New England Biolabs).

    Techniques: Luciferase, Expressing, Activity Assay, Standard Deviation