E. coli genome modification for metabolic engineering, subcloning approaches, plasmid modification, mutations
Recombineering refers to recombination-mediated genetic engineering and specifies a well-defined and precise modification of DNA. This outstanding technology was developed by the group of A. Francis Stewart at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany in the late 1990s.
Strain modifications examples
The pgl gene was successfully integrated into the chromosome of the pgl deficient E. coli expression strain (E. coli T7E1, please see above). The corresponding PGL enzyme catalyzes the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconate and thus prevents the accumulation of the electrophile substrate. A loss of the enzyme activity will result in random gluconoylation of cellular proteins including recombinant ones. The...
An efficient pathway engineering of E. coli was performed by several targeted gene knock-outs using Red/ET recombination. Overall five E. coli strains with up to seven knock-outs were generated. The most promising strain showed an improved succinate production of up to 47%in comparison to the wildtype (4%).
Voir tous les Kits de recombinaison Red/ET :
Red/ET recombination has been used for a promotor fine tuning in E. coli by replacing an entire promotor region or 5´elements with a synthetic promotor library (SPL). SPL is based on randomized sequences flanking the consensus -10 and -35 promotor regions and allows fine-tuning of bacterial gene expression, overcoming the common all-or-nothing approach: change of gene expression by deletion and/or strong overexpression...
