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Team:SCAU-China/nadE-overexpression
From 2014.igem.org
Overexpression of nadE
Electricity active cells (EAC) generate electrons by organic substrate metabolism and transfer them through extracellular transport to an electrode surface in Microbial fuel cell (MFCs). Therefore, genetic modifications that increase the amount of electrons in EAC is one of feasibilities to optimize the electricity power output of MFCs.
Recent studies have proved that the intracellular redox state of EAC is one of the most important physiological traits of extracellular electron transfer efficiency.1 Particularly, the NAD+(H) pool size plays a central role of most metabolic pathways. In this study, we aim that, overexpression of gene nadE which encodes a NAD synthetase and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), may increase the NAD+ level. Therefore the augmented pool size of NAD+(H) result in promotion of NADH (the carrier of electrons) level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC.
In our project, K1373001 and K1373002 are two new parts for over-expression of nadE gene, based on two existing biobricks K608002 (strong constitutive promoter with strong RBS) and K608010 (weak constitutive promoter with Strong RBS), respectively. We confirmed the mRNA and protein expression level of NAD synthetase in corresponding genetically modified strains with BBa_K1373001 or BBa_K1373002 by semi-quantitative RT-PCR and SDS-PAGE assay. [For more results]
Recent studies have proved that the intracellular redox state of EAC is one of the most important physiological traits of extracellular electron transfer efficiency.1 Particularly, the NAD+(H) pool size plays a central role of most metabolic pathways. In this study, we aim that, overexpression of gene nadE which encodes a NAD synthetase and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), may increase the NAD+ level. Therefore the augmented pool size of NAD+(H) result in promotion of NADH (the carrier of electrons) level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC.
Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis
In our project, K1373001 and K1373002 are two new parts for over-expression of nadE gene, based on two existing biobricks K608002 (strong constitutive promoter with strong RBS) and K608010 (weak constitutive promoter with Strong RBS), respectively. We confirmed the mRNA and protein expression level of NAD synthetase in corresponding genetically modified strains with BBa_K1373001 or BBa_K1373002 by semi-quantitative RT-PCR and SDS-PAGE assay. [For more results]
