    2.1.1.107 | analysis |
uroporphyrinogen III methyltransferase (UMT) is a reporter owing to the catalytic products in the cells that emit strong red fluorescence under UV light. The gene encoding the functional barley UMT (bUMT) is engineered by error-prone PCR and the application of UMT as a red fluorescent reporter in Escherichia coli is broadened. A variant, termed mbUMT, is selected and emitts stronger cell fluorescence than the wild type bUMT expressed in different Escherichia coli strains, under different promoters and induction conditions respectively. The constructed mbUMT with a C-terminal ssrA tag is degraded in cells by the protease ClpXP encoded by Escherichia coli chromosome, whereas the bUMT is expressed as active aggregates. Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence. The results suggest that the evolved bUMT is a better candidate to monitor in vivo degradation by Escherichia coli ClpXP |
747676 |
