Difference between revisions of "Part:BBa K4169029"

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We cultivated E. coli BL21 containing tmd, V344C tmd and E. coli BL21 without tmd (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 three tubes of bacteria and making them almost have no difference, we added some TMA into bacteria cultures to make the concentration of substrate TMA 5×10-5mol/L and continued to cultivate them. Take samples before we add TMA, and add TMA for 0 min, 10 min, 20min, 3h, 6h, 9h.
 
We cultivated E. coli BL21 containing tmd, V344C tmd and E. coli BL21 without tmd (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 three tubes of bacteria and making them almost have no difference, we added some TMA into bacteria cultures to make the concentration of substrate TMA 5×10-5mol/L and continued to cultivate them. Take samples before we add TMA, and add TMA for 0 min, 10 min, 20min, 3h, 6h, 9h.
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This is how we handle bacteria samples.
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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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<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>
 
<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>
 
<center><b>Figure 2.</b> Concentration Changes of Metabolism Substrate DMA </center>
 
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Results show that expressed TMADH and V344C could metabolize TMA into DMA successfully, while V344C is more efficient.
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===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 15:32, 12 October 2022


Mutated TMADH

After expressing, it'll produce trimethylamine dehydrogenase (TMADH (EC 1.5.99.7)). The enzyme TMADH is an iron–sulfur flavoprotein which catalyses the oxidative demethylation of trimethylamine (TMA) to dimethylamine and formaldehyde:
(CH3)3N + H2O → (CH3)2NH + CH2O +2H+ + 2e-.

But its sequence is different from wide type. Amino acid 344 is mutated from Val to Cys. The V334C mutant is designed for wiring from the protein close to the 4Fe–4S centre, but the initial electron transfer is activated on the opposite side of the protein, close to the FMN prosthetic group, by substrate (trimethylamine) binding at the active site.[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 trimethylamine metabolism in bacteria.


Protein Molecular Structures


Figure 2.Protein molecular structures of trimethylamine dehydrogenase and it's mutated site V344C.


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 tmd. tmd is induced E. coli BL21 with tmd.



We cultivated E. coli BL21 containing tmd, V344C tmd and E. coli BL21 without tmd (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 three tubes of bacteria and making them almost have no difference, we added some TMA into bacteria cultures to make the concentration of substrate TMA 5×10-5mol/L and continued to cultivate them. Take samples before we add TMA, and add TMA for 0 min, 10 min, 20min, 3h, 6h, 9h.


Figure 2. Concentration Changes of Metabolism Substrate DMA

Results show that expressed TMADH and V344C could metabolize TMA into DMA successfully, while V344C is more efficient.

===Sequence and Features=== Sequence and Features BBa_K4169029 SequenceAndFeatures