Difference between revisions of "Part:BBa K4169029"
m (→Engineering Success) |
|||
(12 intermediate revisions by the same user not shown) | |||
Line 3: | Line 3: | ||
<partinfo>BBa_K4169029 short</partinfo> | <partinfo>BBa_K4169029 short</partinfo> | ||
− | + | 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>. | |
+ | <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== | ||
+ | <p> | ||
+ | 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> | ||
− | < | + | <html> |
− | === | + | <div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/trimethylamine-matabolism/tma-meta.png" style = "width:50%"></center><br></div> |
+ | </html> | ||
− | < | + | <center><b>Figure 1.</b>Pathways for trimethylamine metabolism in bacteria. </center> |
+ | <br> | ||
+ | |||
+ | ==Protein Molecular Structures== | ||
+ | <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> | ||
+ | </html> | ||
+ | |||
+ | <center><b>Figure 2.</b>Protein molecular structures of trimethylamine dehydrogenase and it's mutated site V344C. </center> | ||
+ | <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=== | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K4169029 SequenceAndFeatures</partinfo> | <partinfo>BBa_K4169029 SequenceAndFeatures</partinfo> | ||
Line 14: | Line 54: | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
+ | |||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4169029 parameters</partinfo> | <partinfo>BBa_K4169029 parameters</partinfo> | ||
<!-- --> | <!-- --> |
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