An eYFP Reporter Gene for the Yeast Two-hybrid System

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

Direct and indirect use of GFP whole cell biosensors for the assessment of bioprocess performances: design of milliliter scale-down bioreactors

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 ▲]

Development of mini scale-down platform based on the response of GFP microbial biosensors

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 ▲]

Interactomic map of the Ets factors family : Identification of unexpected functions in mRNA processing

Poster (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 ▲]

Interaction of HTLV-1 Tax with minichromosome maintenance proteins accelerates the replication timing program

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 ▲]

Potentiality 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

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 ▲]

Establishment of an interactomic map of the Ets factors family: Towards a better understanding of their roles in oncogenic processes

Poster (2011, April 29)

Ets transcription factors have been involved in several cancers such as leukemia, prostate cancer and Ewing’s sarcoma. They regulate the expression of genes controlling important biological processes such ... [more ▼]

Ets transcription factors have been involved in several cancers such as leukemia, prostate cancer and Ewing’s sarcoma. They regulate the expression of genes controlling important biological processes such as cellular proliferation, differentiation, apoptosis, metastasis, and transformation. This family of transcription factors is characterized by its highly conserved DNA-binding domain called the ETS domain and members are classified into subfamilies based on sequence homology criterion. We built a protein-protein interaction (PPI) network of the 27 Ets proteins and of their individual functional domains using a high-throughput yeast-two hybrid (Y2H) screening method. That Y2H network was expanded with confident literature-curated PPIs to obtain a comprehensive Ets interaction network. By considering connectivity between Ets interaction partners, we were able to segregate highly connected clusters of proteins from that network. Analysis of ontologies enrichment of those clusters enabled to confirm well-established roles and regulations of Ets factors, but also to suggest new ones. Biological validation of one precise cluster could be used as a rule of a thumb to globally confirm the bioinformatic analysis of our Ets PPI network and the potential physiological or pathological roles and regulation of Ets factors. [less ▲]

Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

in Biotechnology Journal (2011), 6

Establishment of an interactomic map of the Ets factors family: Towards a better understanding of their roles in oncogenic processes

Poster (2010, March)

Ets transcription factors play key roles in several cancers such as leukemia, prostate cancer and Ewing’s sarcoma. They regulate the expression of genes controlling biological processes such as cellular ... [more ▼]

Ets transcription factors play key roles in several cancers such as leukemia, prostate cancer and Ewing’s sarcoma. They regulate the expression of genes controlling biological processes such as cellular proliferation, differentiation, apoptosis, metastasis, and transformation. This family is characterized by a highly conserved DNA-binding domain (ETS domain) and is classified into subfamilies according to sequence homology between the members. Using a high-throughput yeast two-hybrid (Y2H) method, we tested the interaction of the major splicing variants of the 28 human Ets factors and their functional domains of interest against the last available version of the human ORFeome (hORFeome v5.1). This screen has identified more than 200 new partners of Ets proteins. Further validation of these new interactions together with previously described interactions will enable a global evaluation of the regulation, and normal and cancerous roles of Ets factors. [less ▲]

Protein-protein interactions and gene expression regulation in HTLV-1 infected cells.

in Frontiers in Bioscience : A Journal and Virtual Library (2009), 14

Human T-cell leukemia virus type 1 (HTLV-1) propagates in CD4 or CD8 T cells and, after extended latency periods of 30-50 years, causes a rapidly fatal leukemia called adult T-cell leukemia/lymphoma (ATL ... [more ▼]

Human T-cell leukemia virus type 1 (HTLV-1) propagates in CD4 or CD8 T cells and, after extended latency periods of 30-50 years, causes a rapidly fatal leukemia called adult T-cell leukemia/lymphoma (ATL). Infection with HTLV-1 is also associated with a degenerative neuromuscular disease referred to as tropical spastic paraparesis or HTLV-1-associated myelopathy. HTLV genome, in addition to the structural proteins and retroviral enzymes, codes for a region at its 3' end originally designated pX. The products of this region (Tax, Rex, p12I, p13II, p30II and HBZ) play important roles in deregulation of cellular functions by either directly disrupting cellular factors or altering transcription of viral and cellular genes. Here, we will review current knowledge of protein-protein interactions that regulate transcriptional functions of proteins encoded by the pX region. [less ▲]