Difference between revisions of "Part:BBa K4169016"

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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.
 
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.
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This is how we handle bacteria samples.
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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 and more details can be seen in the parts experience page.
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700 µl bacteria samples were centrifugated at 3000 × g 5 min at 4 °C, take 500µl supernatant. Then 300 µl freshly prepared 10 mM solution of FMOC-Cl in acetonitrile was added, after 1 min, 100 µl 100 mM glycine solution was added to neutralize the reaction.
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This is our method of HPLC
 
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Supernatant was transferred to new tube for analysis on HPLC system.
 
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10 µl was loaded on to C18 column equilibrated with acetonitrile-buffer (50%) at flow rate 0.75 ml/min. The column was then flushed with a gradient to 100% elutant buffer B (acetonitrile 75% v/v) within 5 min. Ultraviolet absorption of column elutant was monitored (220 nm) and DMA quantification was calculated based on ratio to standard sample peak area.
 
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Results shows that although DMATH can be expressed, we can't confirm it could work in E. coli BL21.
 
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<div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/hplc-substance.png" style = "width:50%"></center><br></div>
 
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<center><b>Figure 2.</b> Concentration Changes of Metabolism Substrate DMA </center>
 
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===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 05:52, 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.


Figure 1.Pathways for dimethylamine metabolism in bacteria.


Protein Molecular Structures

Dimethylamine dehydrogenase(DMADH) exist as dimers.


Figure 2.Protein molecular structures of dimethylamine dehydrogenase.


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.


Figure 1. Control is E. coli BL21 without dmd. dmd is induced E. coli BL21 with dmd.



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 and more details can be seen in the parts experience page.

Sequence and Features

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 897
    Illegal AgeI site found at 1291
  • 1000
    COMPATIBLE WITH RFC[1000]