References of "Twizere, Jean-Claude"
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See detailTARGETS FOR RETROVIRUS ASSOCIATED DISEASES
Twizere, Jean-Claude ULg

Patent (in press)

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See detailScale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according substrate heterogeneities
Brognaux, Alison ULg; Francis, Frédéric ULg; Twizere, Jean-Claude ULg et al

in Bioprocess and Biosystems Engineering (2014)

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane ... [more ▼]

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high-cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64%) confirming the fact that leakage is involved in high-cell density cultures. About 50% of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by 2D-DIGE analysis (2-Dimensional Differential in-Gel Electrophoresis) and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant. [less ▲]

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See detailScale-down effect on the extracellular proteome of Escherichia coli: correlation with membrane permeability and modulation according substrate heterogeneities
Brognaux, Alison ULg; Francis, Frédéric ULg; Twizere, Jean-Claude ULg et al

in Bioprocess and Biosystems Engineering (2014)

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane ... [more ▼]

Protein leakage is induced in well-mixed fed-batch bioreactor by comparison with cultures carried out in scale-down conditions. This effect is attributed to a progressive increase of cell membrane permeability and the synthesis of several outer-membrane components allowing to cope with substrate limitation commonly found in high-cell density culture. A comparative analysis of protein leakage has thus been performed in well-mixed bioreactors and in scale-down devices. The extracellular proteome of E.coli has been investigated by 2D-gel electrophoresis and identified by subsequent MALDI-TOF analysis. On 110 picked spots, 67 proteins have been identified and the sub-localisation and the molecular function of these proteins have been determined. A majority of the extracellular proteome was composed of outer-membrane and periplasmic proteins (64%) confirming the fact that leakage is involved in high-cell density cultures. About 50% of this extracellular proteome was composed of transport and binding proteins. Furthermore, the more abundant spots on the gel corresponded to porin proteins and periplasmic transporters. In particular, the OmpC porin was found to be very abundant. Moreover, the scale-down effect on this extracellular proteome has been investigated by 2D-DIGE analysis (2-Dimensional Differential in-Gel Electrophoresis) and significant differences have been observed by comparison with culture carried out in well-mixed systems. Indeed, since substrate limitation signal is alleviated in this kind of apparatus, cell permeability was lowered as shown by flow cytometry. In scale-down conditions, protein leakage was thus less abundant. [less ▲]

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See detailAn interaction map for HTLV-1 Tax and PDZ-containing proteins.
Blibek, Karim ULg; Rambout, Xavier ULg; beaufays, Jérôme et al

Poster (2013, June 29)

Human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes for the Tax protein, which has a transforming capacity in vitro. Tax contains at its C-terminus a binding motif for PDZ domain-containing ... [more ▼]

Human T-cell leukemia virus type 1 (HTLV-1) retrovirus encodes for the Tax protein, which has a transforming capacity in vitro. Tax contains at its C-terminus a binding motif for PDZ domain-containing proteins (PSD95-DLG1-ZO1). It has been shown that the C-terminal motif of Tax is involved in Tax oncogenic capacity. Ten different PDZ domain-containing proteins have been reported to interact with Tax, but the specificity of Tax-human PDZome interactions has not been investigated. The objective of this study is to obtain a comprehensive interactome map for Tax and the human PDZome and to determine a global role of Tax-PDZ interactions in HTLV-1 biology. [less ▲]

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See detailInhibition of Tax transformation activity using a small molecule targetting Tax/PDZ domain interactions.
Blibek, Karim ULg; Fujii, Naoaki; Legros, Sebastien et al

Poster (2013, June 29)

Primate T-lymphotropic virus species comprise four members (HTLV-1 to -4) that have been discovered in human. Only the HTLV-1 infection leads to adult T-cell leukemia/lymphoma (ATLL) and tropical spastic ... [more ▼]

Primate T-lymphotropic virus species comprise four members (HTLV-1 to -4) that have been discovered in human. Only the HTLV-1 infection leads to adult T-cell leukemia/lymphoma (ATLL) and tropical spastic paraparesis (TSP), an immune degenerative neurologic syndrome. All the four viruses share a similar genomic organization and encode transforming Tax oncoproteins. In contrast to HTLV-2 and 4, HTLV-1 and 3 Tax proteins contain a PSD-95/Drosophila Discs Large/Zona Occludens-I (PDZ) binding motif at their C-terminal that has been shown to play crucial roles in the distinct transforming properties of the Tax proteins. To systematically investigate PDZ-containing proteins roles in HTLV-1 biology, we initiated a global interactome network analysis of Tax and associated human PDZ-containing proteins. This was accomplished through the use of our framework of binary interactome mapping that includes stringent yeast two hybrid and pulldown screening, systematic retesting by protein complementation assay and evaluation of PDZ gene expression in T lymphocytes. [less ▲]

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See detailAn eYFP Reporter Gene for the Yeast Two-hybrid System
Damon, Coralie; Boxus, Mathieu; Twizere, Jean-Claude ULg et al

in Protein Journal (2013), 32(2), 126-130

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See detailInteractomic map of the Ets factors family : Identification of unexpected functions in mRNA processing
Rambout, Xavier ULg; Simonis, Nicolas; Brohée, Sylvain et al

Poster (2013, January 28)

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets ... [more ▼]

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets factors in order to better define their roles and regulations in normal and oncogenic processes. The Ets interactome was built on a high-throughput yeast-two hybrid (Y2H) approach, and a literature and database curation. We identified 431 PPIs and 276 different protein partners. Clustering of the Ets interactome divided it into 24 functional subnetworks classified on their novelty index and their size. Cluster#1 was exclusively composed of newly identified interaction partners and was highly connected to the Erg subfamily of Ets factors. Gene ontology enrichment analysis revealed that it was associated to mRNA processing. In support of this result, we observed in HeLa cells that ERG and the components of cluster#1 localized in p-bodies and stress granules, physically linked cytoplasmic sites of mRNA degradation and silencing. Hence, we hypothesized that Erg proteins might have a role in post-transcriptional gene regulation and be involved in cellular mRNAs degradation. To test this hypothesis, we performed a MS2-based tethering assay and showed that the recruitment of ERG on a mRNA reporter promoted inhibition of its expression via a two-fold decrease of its half-life. ERG controls degradation of target mRNAs via different mechanisms including polysome stability, mRNA deadenylation, and p-bodies aggregation. A microarray-based appraoch identified 321 endogeneous genes whose mRNA decay rate was lowered in ERG silenced cells. Results point out the Nter domain of ERG as the predominant domain required for mRNA degradation. Importantly, oncogenic TET-Erg fusions described in AML and Ewing’s sarcoma exhibited diminished ability to degrade target mRNAs, concomitantly with the loss of the ERG Nter domain. This reinforces the important role of Erg proteins in mRNA degradation in cancer. [less ▲]

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See detailThe homeodomain transcription factor Hoxa2 interacts with and promotes the proteasomal degradation of the E3 ubiquitin protein ligase RCHY1.
Bergiers, Isabelle; Bridoux, Laure; Nguyen, Nathan et al

in PloS one (2013), 8(11), 80387

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of ... [more ▼]

Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53. [less ▲]

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See detailPP2A regulatory subunit Balpha controls endothelial contractility and vessel lumen integrity via regulation of HDAC7.
Martin, Maud ULg; Geudens, Ilse; Bruyr, Jonathan et al

in EMBO Journal (2013)

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens ... [more ▼]

To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Balpha regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Balpha in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Balpha in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Balpha controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Balpha, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Balpha/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion. [less ▲]

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See detailDirect and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: design of milliliter scale-down bioreactors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Biotechnology Progress (2013), 29(1), 48-59

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional ... [more ▼]

Substrate limitation responsive biosensors have been used for the development of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e., uspA::gfp, csiE::gfp and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response to substrate limitation and are significantly repressed when cultures are carried out in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini-scale-down platform is proposed as a high throughput tool to rapidly investigate the usefulness of a given microbial biosensor. This platform is composed of shake flasks able to operate in fed-batch mode either using the slow release or the intermittent feeding principle. Local heterogeneities were reproduced at the level of these mini-bioreactors (operating under the intermittent feeding principle) and caused a decrease in GFP expression as in conventional scale-down reactors. The presence of GFP in supernatants was also noted and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with membrane permeability. [less ▲]

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See detailDevelopment of mini scale-down platform based on the response of GFP microbial biosensors
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Poster (2012, May 18)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In our previous works, green fluorescent protein reporters have been used as biosensors of the heterogeneities generated in a two compartment scale-down reactor. As there is a huge variety of available whole cell biosensor to characterize the impact of such heterogeneities at the biological level, there is a need for high-throughput cultivation tools in order to investigate the usefulness of a given microbial biosensor among a library comprising several thousands of clones. This work is based on this statement and aims to investigate the potentialities of a mini scale-down platform. Four green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli: rpoS::gfp, uspA::gfp, csiE::gfp and yciG::gfp. The promoters rpoS and uspA are induced in response to a variety of stresses whereas the two other promoters, csiE and yciG, are supposed to be more specific in front of a glucose limitation. First, the response of these biosensors has been assessed in chemostat reactors. These kinds of experiments allow easier interpretation of responses of stress gene related to a glucose limitation since the extracellular conditions are constants and cells are renewed. Biosensors carrying the csiE and yciG promoters have exhibited an induction in function of the glucose limitation. Secondly, a scale-down platform has been tested with the same biosensors and two kinds of glucose addition mode. This scale-down platform involves high-throughput cultivation tools, i.e. in our case shake flask, equipped with non-invasive optical sensors for the monitoring of the dissolved oxygen profile in front of the glucose addition mode. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. The intermittent addition of glucose induces a slow down at the level of the GFP synthesis, suggesting that temporal accumulation of glucose inhibits the activity of the yciG and csiE promoters. In conclusion, the scale-down platform is able to reproduce the same kind of glucose fluctuations that encounters the cells in large-scale processes but not allows studying the impact of high-cell density culture on gene expression. [less ▲]

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See detailUse of microbial biosensors to detect substrate heterogeneities at the single cell level and assess microbial viability: Validation of a mini-bioreactor platform
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

Conference (2012, March 15)

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor ... [more ▼]

The basic principle adopted in our studies is to use substrate limitation responsive biosensors in order to detect spatial glucose heterogeneities inside industrial bioreactors (whole-cell biosensor). Indeed, such heterogeneities cause a lowering of the biomass yield and an increase of by-products concentration. In this work, we have used these biosensors for the elaboration of a mini-bioreactor platform that can be used as a scale-down tool. Three green fluorescent protein (GFP) transcriptional reporters have been chosen in Escherichia coli, i.e. uspA::gfp, csiE::gfp and yciG::gfp. Our previous studies have shown that these kinds of promoters are induced in response of substrate limitation and exhibit a strong fluorescence attenuation when cultivated in heterogeneous bioreactors. This sensitivity to substrate limitation has been confirmed in the case of the csiE and yciG biosensors. A mini scale-down platform has been proposed as a high throughput tool to investigate rapidly the usefulness of a given microbial biosensor. This platform is composed of shake flask able to operate in fed-batch mode either by using the slow release or the intermittent feeding principle. The first system is based on a commercial package (Enbase) based on the enzymatic release of glucose in the medium. The Enbase system allows the generation of a very smooth glucose profile without any perturbations. For comparison purpose, we have also used an intermittent feeding that induces strong fluctuation at the level of the glucose and the dissolved oxygen concentration. Local heterogeneities have thus been reproduced at the level of these mini-bioreactors and these one have caused a decrease of GFP expression, as in conventional scale-down reactor. The presence of GFP in supernatants has also been noticed and seems to be correlated with the substrate limitation signal for the three cultivation systems considered in this work (i.e., chemostat, conventional and mini-bioreactors) and with the membrane permeability. [less ▲]

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See detailInteractomic map of the Ets factors family : Identification of unexpected functions in mRNA processing
Rambout, Xavier ULg; Simonis, Nicolas; Demoitié, Pauline et al

in Keystone symposium - Protein-RNA Interactions in Biology and Disease (C1) (2012, March 05)

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain, the ETS domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the ... [more ▼]

The Ets factors are a family of 27 transcription factors characterized by their unique DNA-binding domain, the ETS domain. We aimed at building a protein-protein interaction (PPI) map (interactome) of the human Ets factors in order to better define their roles and regulations in normal and oncogenic processes. The Ets interactome was built on a high-throughput yeast-two hybrid (Y2H) approach, and a literature and database curation of confident interactions which led us to the identification of 602 PPIs and 369 different protein partners. Clusterization using the Network Analysis Tool box (NeAT) divided the ETS interactome into 39 functional sub-networks. Among these, we identified Cluster16 as highly connected to the Erg ETS subfamily. A gene ontology (GO) enrichment analysis revealed that Cluster16 was associated to various aspects of mRNA processing. We therefore hypothesized that Erg factors might have a role in post-transcriptional gene regulation. This would constitute a entirely new and undisclosed role for ETS factors, which are so far firmly established as transcription factors. In support of our hypothesis, we observed that ERG localized in p-bodies, cytoplasmic sites of mRNA decay. Interestingly, under various cellular stresses, a portion of ERG and its partners from Cluster16 localized in stress granules, cytoplasmic sites of mRNA silencing physically linked to p-bodies. Hence, we hypothesized that Erg proteins might be involved in cellular mRNAs degradation. To test this, we performed a MS2-based tethering assay and showed that the recruit-ment of Erg factors promoted degradation of a reporter mRNA, mainly via its N-ter domain. Very importantly, oncogenic TET-Erg fusions described in AML and Ewing’s sarcoma exhibited diminished ability to degrade target mRNAs, concomitantly with the loss of the N-ter domain of the corresponding Erg protein. This re-inforces the important role of Erg proteins in mRNA degradation in cancer. Our efforts are now concentrated on identifying the molecular determinants behind this new function of Erg proteins. [less ▲]

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See detailInteraction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program
Boxus, Mathieu ULg; Twizere, Jean-Claude ULg; Legros, Sébastien et al

in Blood (2012), 119

The Tax oncoprotein encoded by the Human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in viral persistence and pathogenesis. HTLV-1 infected cells proliferate faster than normal lymphocytes ... [more ▼]

The Tax oncoprotein encoded by the Human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in viral persistence and pathogenesis. HTLV-1 infected cells proliferate faster than normal lymphocytes, expand through mitotic division and accumulate genomic lesions. Here, we show that Tax associates with the minichromosome maintenance MCM2-7 helicase complex and localizes to origins of replication. Tax modulates the spatiotemporal program of origin activation and fires supplementary origins at the onset of S phase. Thereby, Tax increases the DNA replication rate, accelerates S phase progression but also generates a replicative stress characterized by the presence of genomic lesions. Mechanistically, Tax favors p300 recruitment and histone hyperacetylation at late replication domains advancing their replication timing in early S phase. [less ▲]

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See detailHost-pathogen interactome mapping for HTLV-1 and -2 retroviruses.
Simonis, Nicolas; Rual, Jean-Francois; Lemmens, Irma et al

in Retrovirology (2012), 9

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL ... [more ▼]

BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) and type 2 both target T lymphocytes, yet induce radically different phenotypic outcomes. HTLV-1 is a causative agent of Adult T-cell leukemia (ATL), whereas HTLV-2, highly similar to HTLV-1, causes no known overt disease. HTLV gene products are engaged in a dynamic struggle of activating and antagonistic interactions with host cells. Investigations focused on one or a few genes have identified several human factors interacting with HTLV viral proteins. Most of the available interaction data concern the highly investigated HTLV-1 Tax protein. Identifying shared and distinct host-pathogen protein interaction profiles for these two viruses would enlighten how they exploit distinctive or common strategies to subvert cellular pathways toward disease progression. RESULTS: We employ a scalable methodology for the systematic mapping and comparison of pathogen-host protein interactions that includes stringent yeast two-hybrid screening and systematic retest, as well as two independent validations through an additional protein interaction detection method and a functional transactivation assay. The final data set contained 166 interactions between 10 viral proteins and 122 human proteins. Among the 166 interactions identified, 87 and 79 involved HTLV-1 and HTLV-2 -encoded proteins, respectively. Targets for HTLV-1 and HTLV-2 proteins implicate a diverse set of cellular processes including the ubiquitin-proteasome system, the apoptosis, different cancer pathways and the Notch signaling pathway. CONCLUSIONS: This study constitutes a first pass, with homogeneous data, at comparative analysis of host targets for HTLV-1 and -2 retroviruses, complements currently existing data for formulation of systems biology models of retroviral induced diseases and presents new insights on biological pathways involved in retroviral infection. [less ▲]

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See detailInteraction network of antimicrobial peptides of Arabidopsis thaliana, based on hith-throughput yeast two-hybrid screening
Damon, Coralie ULg; Dmitrieva, Joelia Borisnova; Muhovski, Yordan et al

in Plant Physiology & Biochemistry (2012)

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See detailPotentiality of using microbial biosensors for the detection of substrate heterogeneities and the assessment of microbial viability in industrial bioreactors: a complete set of experiments in chemostat and scale-down reactors, and elaboration of a mini scale-down platform
Brognaux, Alison ULg; Neubauer, Peter; Twizere, Jean-Claude ULg et al

in Communications in Agricultural and Applied Biological Sciences (2012), 77(1), 3-7

Substrate limitation responsive biosensors have been used in order to detect spatial substrate heterogeneities, , inside industrial bioreactors (whole-cell biosensor). Three green fluorescent protein (GFP ... [more ▼]

Substrate limitation responsive biosensors have been used in order to detect spatial substrate heterogeneities, , inside industrial bioreactors (whole-cell biosensor). Three green fluorescent protein (GFP) transcriptional reporters have been chosen in E.coli, i.e. uspA::gfp, csiE::gfp and yciG::gfp. The promoter uspA is induced in response to a variety of stresses whereas the two other promoters, csiE and yciG, are supposed to be more specific in front of a substrate limitation. The responsiveness of these biosensors has been assessed in chemostat reactor. Secondly, the same biosensors have been tested in well-mixed laboratory reactors and in scale-down reactors able to reproduce industrial conditions. Finally, a mini scale-down platform has been proposed as a high throughput tool to investigate rapidly the usefulness of a given microbial biosensor. Local heterogeneities in mini-bioreactor have caused a decrease of GFP expression, as in scale-down reactor. The presence of GFP in supernatants was noticed and this leakage seems to be correlated with the membrane permeability. [less ▲]

Detailed reference viewed: 84 (27 ULg)