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Applications - Metabolic engineering
Metabolic screen: principle, implementation and validation
In an initial stage, a metabolic screen was designed and constructed. The principle of a metabolic screen consists in making bacterial growth depend on the product of the reaction characterized by the enzyme one seeks to improve or by the metabolic pathway subjected to evolution. In the absence of substrate limitation, the availability of the reaction product to the host cells depends on the effectiveness of the conversion catalyzed. The robustness of the genetic selection, i.e. its stability over time and under various culture conditions, and the strict dependence on the gene or genes selected constitute fundamental prerequisites.
The usual host microorganism is
Escherichia coli. The genetic modifications necessary for construction of the metabolic screen are inserted by the conventional methods of molecular genetics.
Evolution and in vivo selection of improved variants
Numerous methods of
in vitro selection of enhanced enzymes are currently available and generally use random mutagenesis protocols for the genes and high-throughput functional screening.
In vivo selection is an alternative to those methods, which are frequently onerous in terms of equipment and labor.
In vivo selection is based on the spontaneous generation of mutations in the genome of large bacterial populations (> 109) cultured in suspension. Selection of the desired variants among the mutants in the pool is implemented by imposing strict culture conditions under which the mutations of interest confer on the mutant bacteria a higher growth rate. In order to select a cell line bearing adaptation mutations in their genome, the cell culture suspension must be maintained for prolonged durations. Genoscope employs a continuous culture system for microorganisms, GM3. The latter automates the prolonged culture stages – dilution of the cell suspension to create selective pressure, regular cleaning of the incubation chamber – and enables various selective regimens to be programmed.
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