|Reference : Development of mini scale-down platform based on the response of GFP microbial biosensors|
|Scientific congresses and symposiums : Poster|
|Life sciences : Biotechnology|
|Development of mini scale-down platform based on the response of GFP microbial biosensors|
|Brognaux, Alison [Université de Liège - ULg > Département des sciences de la vie > Biochimie et microbiologie industrielles >]|
|Neubauer, Peter [ > > ]|
|Twizere, Jean-Claude [Université de Liège - ULg > Chimie et bio-industries > Biologie cell. et moléc. >]|
|Thonart, Philippe [Université de Liège - ULg > Département des sciences de la vie > Biochimie et microbiologie industrielles >]|
|Delvigne, Frank [Université de Liège - ULg > Chimie et bio-industries > Bio-industries >]|
|du 15 au 18 mai 2012|
|[en] whole cell biosensors ; mini-bioreactors ; substrate heterogeneities|
|[en] 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.
|Fonds pour la formation à la Recherche dans l'Industrie et dans l'Agriculture (Communauté française de Belgique) - FRIA|
|Researchers ; Professionals ; Students|
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