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Michiel Kleerebezem - One of the best experts on this subject based on the ideXlab platform.

  • 10 years of the nisin-controlled Gene Expression System (NICE) in Lactococcus lactis
    Applied Microbiology and Biotechnology, 2005
    Co-Authors: Igor Mierau, Michiel Kleerebezem
    Abstract:

    Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of Genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled Gene Expression System, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago—the NIsin Controlled Gene Expression System, called NICE. This System has become one of the most successful and widely used tools for regulated Gene Expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis , the fundaments, components and function of the NICE System. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-Expression of homologous and heterologous Genes for functional studies and to obtain large quantities of specific Gene products, (2) metabolic engineering, (3) Expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) Expression of Genes with toxic products and analysis of essential Genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.

  • Controlled Gene Expression Systems for lactic acid bacteria: transferable nisin-inducible Expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp.
    Applied and environmental microbiology, 1997
    Co-Authors: Michiel Kleerebezem, Elaine E. Vaughan, W.m. De Vos, Marke M. Beerthuyzen, Oscar P. Kuipers
    Abstract:

    A transferable dual-plasmid inducible Gene Expression System for use in lactic acid bacteria that is based on the autoregulatory properties of the antimicrobial peptide nisin produced by Lactococcus lactis was developed. Introduction of the two plasmids allowed nisin-inducible Gene Expression in Lactococcus lactis MG1363, Leuconostoc lactis NZ6091, and Lactobacillus helveticus CNRZ32. Typically, the beta-glucuronidase activity (used as a reporter in this study) remained below the detection limits under noninducing conditions and could be raised to high levels, by addition of subinhibitory amounts of nisin to the growth medium, while exhibiting a linear dose-response relationship. These results demonstrate that the nisin-inducible System can be functionally implemented in lactic acid bacteria other than Lactococcus lactis.

Jane A Langdale - One of the best experts on this subject based on the ideXlab platform.

  • a modular steroid inducible Gene Expression System for use in rice
    BMC Plant Biology, 2019
    Co-Authors: Daniela Vlad, Basel Abujamous, Peng Wang, Jane A Langdale
    Abstract:

    Chemically inducible Systems that provide both spatial and temporal control of Gene Expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR System and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The System is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous Genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). Our results demonstrate that the System is suitable for General use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

  • optimization of a modular steroid inducible Gene Expression System for use in rice
    bioRxiv, 2019
    Co-Authors: Daniela Vlad, Basel Abujamous, Peng Wang, Marketa Samalova, Ian Moore, Jane A Langdale
    Abstract:

    SUMMARY Chemically inducible Systems that provide both spatial and temporal control of Gene Expression are essential tools, with many applications in plant biology. Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR System and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The System is tightly regulated and highly sensitive to DEX application, with six hours of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous Genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). The System is thus suitable for General use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Igor Mierau - One of the best experts on this subject based on the ideXlab platform.

  • 10 years of the nisin-controlled Gene Expression System (NICE) in Lactococcus lactis
    Applied Microbiology and Biotechnology, 2005
    Co-Authors: Igor Mierau, Michiel Kleerebezem
    Abstract:

    Lactococcus lactis is a Gram-positive lactic acid bacterium that, in addition to its traditional use in food fermentations, is increasingly used in modern biotechnological applications. In the last 25 years great progress has been made in the development of Genetic engineering tools and the molecular characterization of this species. A new versatile and tightly controlled Gene Expression System, based on the auto-regulation mechanism of the bacteriocin nisin, was developed 10 years ago—the NIsin Controlled Gene Expression System, called NICE. This System has become one of the most successful and widely used tools for regulated Gene Expression in Gram-positive bacteria. The review describes, after a brief introduction of the host bacterium L. lactis , the fundaments, components and function of the NICE System. Furthermore, an extensive overview is provided of the different applications in lactococci and other Gram-positive bacteria: (1) over-Expression of homologous and heterologous Genes for functional studies and to obtain large quantities of specific Gene products, (2) metabolic engineering, (3) Expression of prokaryotic and eukaryotic membrane proteins, (4) protein secretion and anchoring in the cell envelope, (5) Expression of Genes with toxic products and analysis of essential Genes and (6) large-scale applications. Finally, an overview is given of growth and induction conditions for lab-scale and industrial-scale applications.

  • industrial scale production and purification of a heterologous protein in lactococcus lactis using the nisin controlled Gene Expression System nice the case of lysostaphin
    Microbial Cell Factories, 2005
    Co-Authors: Igor Mierau, Peter Leij, Iris I Van Swam, Barry Blommestein, Esther Floris, James Mond, Eddy J Smid
    Abstract:

    The NI sin-C ontrolled Gene E xpression System NICE of Lactococcus lactis is one of the most widespread used Expression Systems of Gram-positive bacteria. It is used in more than 100 laboratories for laboratory-scale Gene Expression experiments. However, L. lactis is also a micro-organism with a large biotechnological potential. Therefore, the aim of this study was to test whether protein production in L. lactis using the NICE System can also effectively be performed at the industrial-scale of fermentation. Lysostaphin, an antibacterial protein (mainly against Staphylococcus aureus) from S. simulans biovar. Staphylolyticus, was used as a model System. Food-grade lysostaphin Expression constructs in L. lactis were grown at 1L-, 300-L and 3000-L scale and induced with nisin for lysostaphin production. The induction process was equally effective at all scales and yields of about 100 mg/L were obtained. Up-scaling was easy and required no specific effort. Furthermore, we describe a simple and effective way of downstream processing to obtain a highly purified lysostaphin, which has been used for clinical phase I trials. This is the first example that shows that nisin-regulated Gene Expression in L. lactis can be used at industrial scale to produce large amounts of a target protein, such as lysostaphin. Downstream processing was simple and in a few steps produced a highly purified and active enzyme.

J Zale - One of the best experts on this subject based on the ideXlab platform.

  • a high throughput transient Gene Expression System for switchgrass panicum virgatum l seedlings
    Biotechnology for Biofuels, 2010
    Co-Authors: Xinlu Chen, Raymie Equi, Holly L Baxter, Kyle Berk, Jin Han, Sujata Agarwal, J Zale
    Abstract:

    Background Grasses are relatively recalcitrant to Genetic transformation in comparison to certain dicotyledons, yet they constitute some of the most important biofuel crops. Genetic transformation of switchgrass (Panicum virgatum L.) has previously been reported after cocultivation of explants with Agrobacterium and biolistics of embryogenic calli. Experiments to increase transient Gene Expression in planta may lead to stable transformation methods with increased efficiency.

Hirozo Oh-oka - One of the best experts on this subject based on the ideXlab platform.

  • Gene Expression System in green sulfur bacteria by conjugative plasmid transfer.
    PloS one, 2013
    Co-Authors: Chihiro Azai, Jiro Harada, Hirozo Oh-oka
    Abstract:

    Gene transfer and Expression Systems in green sulfur bacteria were established by bacterial conjugation with Escherichia coli. Conjugative plasmid transfer from E. coli S17-1 to a thermophilic green sulfur bacterium, Chlorobaculum tepidum (formerly Chlorobium tepidum) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in C. tepidum cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, C. limnaeum (formerly Chlorobium phaeobacteroides) RK-j-1, was also achieved with pDSK5192. The Expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the pscAB Gene cluster on the C. tepidum genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆cycA and ∆soxB mutants were completely rescued after introduction of pDSK5191-cycA and -soxB that were designed to express their complementary Genes. On the other hand, pDSK5191-6xhis-pscAB, which incorporated the Gene cluster of 6xhis-pscA and pscB, produced approximately four times more of the photosynthetic reaction center complex with His-tagged PscA as compared with that expressed in the genome by the conventional natural transformation method. This Expression System, based on conjugative plasmid, would be applicable to General molecular biological studies of green sulfur bacteria.

  • Gene Expression System in Green Sulfur Bacteria by Conjugative Plasmid Transfer
    2013
    Co-Authors: Chihiro Azai, Jiro Harada, Hirozo Oh-oka
    Abstract:

    Gene transfer and Expression Systems in green sulfur bacteria were established by bacterial conjugation with Escherichia coli. Conjugative plasmid transfer from E. coli S17-1 to a thermophilic green sulfur bacterium, Chlorobaculum tepidum (formerly Chlorobium tepidum) WT2321, was executed with RSF1010-derivative broad-host-range plasmids, named pDSK5191 and pDSK5192, that confer erythromycin and streptomycin/spectinomycin resistance, respectively. The transconjugants harboring these plasmids were reproducibly obtained at a frequency of approximately 10-5 by selection with erythromycin and a combination of streptomycin and spectinomycin, respectively. These plasmids were stably maintained in C. tepidum cells in the presence of these antibiotics. The plasmid transfer to another mesophilic green sulfur bacterium, C. limnaeum (formerly Chlorobium phaeobacteroides) RK-j-1, was also achieved with pDSK5192. The Expression plasmid based on pDSK5191 was constructed by incorporating the upstream and downstream regions of the pscAB Gene cluster on the C. tepidum genome, since these regions were considered to include a constitutive promoter and a ρ-independent terminator, respectively. Growth defections of the ∆cycA and ∆soxB mutants were completely rescued after introduction of pDSK5191-cycA and-soxB that were designed to express their complementary Genes. On the other hand, pDSK5191-6xhis-pscAB, which incorporated the Gene cluster of 6xhis-pscA and pscB, produced approximately four times more of th