Difference between revisions of "Part:BBa K4169016"
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− | + | Unfortunately, at present, we can only verify that our dimethylamine dehydrogenase can be expressed, but we cannot prove that it is active. More work is still in progress. | |
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Revision as of 05:49, 14 October 2022
produce DMADH
After expressing, it'll produce dimethylamine dehydrogenase (DMADH). The enzyme DMADH is an iron–sulfur flavoprotein which catalyses the oxidative demethylation of dimethylamine (DMA) to methylamine and formaldehyde:
(CH3)2N + H2O → CH3NH + CH2O +2H+ + 2e-[1].
Metabolic Pathway
This enzyme is a complex iron-sulfur flavoprotein that transfers electrons to the soluble flavoprotein known as electron transferring flavoprotein[2]. It couldn't work extracellular isolated.
![](https://static.igem.wiki/teams/4169/wiki/backword/dma/trimethylamine-matabolism/dma-meta.png)
Protein Molecular Structures
Dimethylamine dehydrogenase(DMADH) exist as dimers.
![](https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/tmadh1-1.png)
Engineering Success
We performed SDS-PAGE to identify that trimethylamine dehydrogenase can be expressed. Because trimethylamine dehydrogenase (TMADHexist as dimers, the protein molecular weight would double. So, protein molecular weight of TMADH is 164.9kDa.
![](https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/gel-dmd.png)
We cultivated E. coli BL21 containing dmd and E. coli BL21 without dmd (Blank) for about 3 hours (OD600 0.6~0.8). Then they were induced by 4mM theophylline for 9 hours. After adjusting the density of two tubes of bacteria and making them almost have no difference, we added some DMA into bacteria cultures to make the concentration of substrate DMA 5×10-5mol/L and continued to cultivate them. Take samples before we add DMA, and add DMA for 0 min, 10 min, 20min, 3h, 6h, 9h.
Unfortunately, at present, we can only verify that our dimethylamine dehydrogenase can be expressed, but we cannot prove that it is active. More work is still in progress.
Sequence and Features
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 897
Illegal AgeI site found at 1291 - 1000COMPATIBLE WITH RFC[1000]