Difference between revisions of "Part:BBa K4169029"
m |
m (→Engineering Success) |
||
| (7 intermediate revisions by the same user not shown) | |||
| Line 4: | Line 4: | ||
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: <br>(CH<sub>3</sub>)<sub>3</sub>N + H<sub>2</sub>O → (CH<sub>3</sub>)<sub>2</sub>NH + CH<sub>2</sub>O +2H<sup>+</sup> + 2e<sup>-</sup>. | 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: <br>(CH<sub>3</sub>)<sub>3</sub>N + H<sub>2</sub>O → (CH<sub>3</sub>)<sub>2</sub>NH + CH<sub>2</sub>O +2H<sup>+</sup> + 2e<sup>-</sup>. | ||
| − | 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. | + | <p> |
| − | + | 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] | |
| + | </p> | ||
==Metabolic Pathway== | ==Metabolic Pathway== | ||
<p> | <p> | ||
| − | This enzyme is a complex iron-sulfur flavoprotein that transfers electrons to the soluble flavoprotein known as electron transferring flavoprotein. It couldn't work extracellular isolated. | + | 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. |
</p> | </p> | ||
| Line 19: | Line 20: | ||
==Protein Molecular Structures== | ==Protein Molecular Structures== | ||
| − | |||
| − | |||
| − | |||
<html> | <html> | ||
<div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/v344c-com.png" style = "width:75%"></center><br></div> | <div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/v344c-com.png" style = "width:75%"></center><br></div> | ||
| Line 29: | Line 27: | ||
<br> | <br> | ||
| + | ====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. | ||
| + | <html> | ||
| + | <div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/gel-tmd.png" style = "width:50%"></center><br></div> | ||
| + | </html> | ||
| + | <center><b>Figure 1.</b> Control is E. coli BL21 without tmd. tmd is induced E. coli BL21 with tmd. </center> | ||
| + | <br> | ||
| + | |||
| + | |||
| + | 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. | ||
| + | |||
| + | <html> | ||
| + | <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> | ||
| + | <html> | ||
| + | <center><b>Figure 2.</b> Concentration Changes of Metabolism Substrate DMA </center> | ||
| + | <br> | ||
| + | <p> | ||
| + | Results show that expressed TMADH and V344C could metabolize TMA into DMA successfully, while V344C is more efficient. | ||
| + | </p> | ||
===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.
Protein Molecular Structures
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.
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.
Results show that expressed TMADH and V344C could metabolize TMA into DMA successfully, while V344C is more efficient.
===Sequence and Features=== Sequence and Features