Difference between revisions of "Part:BBa K4169029:Experience"

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===Applications of BBa_K4169029===
 
===Applications of BBa_K4169029===
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To reduce the amount of trimethylamine (TMA) in the gut, we considered the degradation of TMA produced by gut microorganisms. So, E. coli containing trimethylamine dehydrogenase gene could catalyse the oxidative demethylation of TMA to dimethylamine and formaldehyde. V344C is a mutated trimethylamine dehydrogenase that is more efficient.
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===User Reviews===
 
===User Reviews===
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====The trimethylamine dehydrogenase by 2022 HZAU-iGEM team====
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Plasmid(4μg) was synthesized by Genscript.
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Firstly, we centrifuged dmd plasmid powder at 5000rpm for 1 min, then added 40μg sterile ddH2O to dissolve it. The plasmid concentration was 100ng/μL. After diluting plasmid solution into10ng/μL, we transformed plasmids into competent cells E. coli BL21. The outcomes of colony PCR is showed below.
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<div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/fig-2.png" style = "width:20%"></center><br></div>
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<center><b>Figure 1.</b> Colony PCR of E. coli BL21containing dmd plasmids. </center>
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<br>
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<p>
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Based on the forecast outcomes of module that modification at position 344 of TMADH has a more profound influence on intra- and inter-molecular electron transport, we mutated Val in position 344 into Cys by. Then we sequenced Sangon Biotech the production of Gel DNA Purification Kit and used it to transform the 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/fig-4.jpg" style = "width:10%"></center><br></div>
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<center><b>Figure 2.</b> Colony PCR of E. coli BL21containing mutated V344C tmd plasmids.</center>
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<br>
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</p>
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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.
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<div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/gel-tmd.png" style = "width:30%"></center><br></div>
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<center><b>Figure 3.</b> Control is E. coli BL21 without tmd. tmd is induced E. coli BL21 with tmd. </center>
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<br>
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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.
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<p>
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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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</p>
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<p>
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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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</p>
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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 4.</b> Concentration Changes of Metabolism Substrate DMA </center>
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<br>
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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>.
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Latest revision as of 07:15, 13 October 2022


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K4169029

To reduce the amount of trimethylamine (TMA) in the gut, we considered the degradation of TMA produced by gut microorganisms. So, E. coli containing trimethylamine dehydrogenase gene could catalyse the oxidative demethylation of TMA to dimethylamine and formaldehyde. V344C is a mutated trimethylamine dehydrogenase that is more efficient.

User Reviews

The trimethylamine dehydrogenase by 2022 HZAU-iGEM team

Plasmid(4μg) was synthesized by Genscript. Firstly, we centrifuged dmd plasmid powder at 5000rpm for 1 min, then added 40μg sterile ddH2O to dissolve it. The plasmid concentration was 100ng/μL. After diluting plasmid solution into10ng/μL, we transformed plasmids into competent cells E. coli BL21. The outcomes of colony PCR is showed below.


Figure 1. Colony PCR of E. coli BL21containing dmd plasmids.


Based on the forecast outcomes of module that modification at position 344 of TMADH has a more profound influence on intra- and inter-molecular electron transport, we mutated Val in position 344 into Cys by. Then we sequenced Sangon Biotech the production of Gel DNA Purification Kit and used it to transform the E. coli BL21.


Figure 2. Colony PCR of E. coli BL21containing mutated V344C tmd plasmids.


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 3. 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.

This is how we handle bacteria samples. 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.

This is our method of HPLC Supernatant was transferred to new tube for analysis on HPLC system. 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.


Figure 4. Concentration Changes of Metabolism Substrate DMA



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