Difference between revisions of "Part:BBa K4169015"

Latest revision as of 08:13, 14 October 2022

produce 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^- [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

Trimethylamine dehydrogenase (TMADH) exist as dimers.

Figure 2.Protein molecular structures of trimethylamine dehydrogenase.

At the same time, we found that the existing structural research on TMADH is relatively thorough through literature review. In combination with the results of mathematical modeling in the optimization of enzyme kinetics, we finally selected the V344C mutant and compared it with the unmutated TMADH to quantitatively detect the enzyme activity.

Experience

We have a more detailed understanding of this enzyme before conducting experiments. The structure of this enzyme is very complex, and the iron sulfur center also makes it difficult to carry out numerical modeling. Of course, some teams tried to express their success before, but failed to fully express their success. This year, our team decided to accept this challenge, trying to express the protein level and using the HPLC method to measure the enzyme activity (the previous studies were all measured by gas chromatography and gas chromatography-mass spectrometry). After about a month, we initially got the ideal result.However, due to the specificity of the enzyme structure, we still hope to design a series of experiments to further verify; But at present, we believe that it can prove that our enzyme can play a role in degradation.

Purpose

We have taken a series verified experiment to figure out whether this dufficult gene can be expressed successfully and can be expressed an active protein successfully then. Although it was some basic molecular cloning research in the early stage, we still made a lot of efforts under the premise of many failures, and finally expressed active TMADH.

The expression at the nucleic acid level

Plasmid(4μg) was synthesized by Genscript. Firstly, we centrifuged tmd 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 BL21 containing tmd plasmids.

The expression at the protein level

We performed SDS-PAGE to identify that trimethylamine dehydrogenase can be expressed. Because trimethylamine dehydrogenase (TMADH exist 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.

Detection of protein activity

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^(-5)mol/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 4. Concentration Changes of Metabolism Substrate DMA

In the first six hours, it can be seen that TMADH successfully metabolized DMA, and the mutant V344C has higher activity. At 9 hours, DMA became less. We speculated that the abnormal metabolism of bacteria might also metabolize dimethylamine as a substrate to other substances.

So the results show that expressed TMADH could metabolize TMA into DMA successfully. Also it can be seen that the mutant has higher activity than normal one, which is in line with the model work.

We also hope that our results will be helpful to future teams and provide them with some information for reference.

References

[1] Jang M H, Basran J, Scrutton N S, et al. The reaction of trimethylamine dehydrogenase with trimethylamine[J]. Journal of Biological Chemistry, 1999, 274(19): 13147-13154. Because trimethylamine dehydrogenase (TMADH) and dimethylamine dehydrogenase (DMADH) exist as dimers

[2] Scrutton N S, Sutcliffe M J. Trimethylamine dehydrogenase and electron transferring flavoprotein[J]. Enzyme-Catalyzed Electron and Radical Transfer, 2000: 145-181.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 388
Illegal PstI site found at 183
Illegal PstI site found at 1782
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 388
Illegal PstI site found at 183
Illegal PstI site found at 1782
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 388
Illegal XhoI site found at 1717
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 388
Illegal PstI site found at 183
Illegal PstI site found at 1782
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 388
Illegal PstI site found at 183
Illegal PstI site found at 1782
Illegal AgeI site found at 879
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4169015 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>.
+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> [1].
 ==Metabolic Pathway==
 <p>
-This enzyme is a complex iron-sulfur flavoprotein that transfers electrons to the soluble flavoprotein known as electron transferring flavoprotein.[1] 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>
@@ Line 27: / Line 27: @@
 <center><b>Figure 2.</b>Protein molecular structures of trimethylamine dehydrogenase. </center>
 <br>
+At the same time, we found that the existing structural research on TMADH is relatively thorough through literature review. In combination with the results of mathematical modeling in the optimization of enzyme kinetics, we finally selected the <i>V344C</i> mutant and compared it with the unmutated TMADH to quantitatively detect the enzyme activity.
+==Experience==
+We have a more detailed understanding of this enzyme before conducting experiments. The structure of this enzyme is very complex, and the iron sulfur center also makes it difficult to carry out numerical modeling. Of course, some teams tried to express their success before, but failed to fully express their success. This year, our team decided to accept this challenge, trying to express the protein level and using the HPLC method to measure the enzyme activity (the previous studies were all measured by gas chromatography and gas chromatography-mass spectrometry). After about a month, we initially got the ideal result.However, due to the specificity of the enzyme structure, we still hope to design a series of experiments to further verify; But at present, we believe that it can prove that our enzyme can play a role in degradation.
+===Purpose===
+We have taken a series verified experiment to figure out whether this dufficult gene can be expressed successfully and can be expressed an active protein successfully then. Although it was some basic molecular cloning research in the early stage, we still made a lot of efforts under the premise of many failures, and finally expressed active TMADH.
+====The expression at the nucleic acid level====
+Plasmid(4μg) was synthesized by Genscript.
+Firstly, we centrifuged tmd 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 <i>E. coli</i> BL21. The outcomes of colony PCR is showed below.
+<html>
+<div class = "center"><center><img src = "https://static.igem.wiki/teams/4169/wiki/backword/dma/tmd-dmd-structure/fig-3.png" style = "width:20%"></center><br></div>
+</html>
+<center><b>Figure 1.</b> Colony PCR of <i>E. coli</i> BL21 containing tmd plasmids. </center>
+<br>
+====The expression at the protein level====
+We performed SDS-PAGE to identify that trimethylamine dehydrogenase can be expressed. Because trimethylamine dehydrogenase (TMADH exist 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 3.</b> Control is <i>E.coli</i> BL21 without <i>tmd</i>.<i>Tmd</i> is induced <i>E. coli</i> BL21 with <i>tmd</i>. </center>
+<br>
+====Detection of protein activity====
+We cultivated <i>E. coli</i> BL21 containing <i>tmd</i>, V344C <i>tmd</i> and <i>E. coli</i> 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^(-5)mol/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///engineering/substance1-2.png" style = "width:50%"></center><br></div>
+</html>
+<center><b>Figure 4.</b> Concentration Changes of Metabolism Substrate DMA </center>
+<br>
+In the first six hours, it can be seen that TMADH successfully metabolized DMA, and the mutant V344C has higher activity. At 9 hours, DMA became less. We speculated that the abnormal metabolism of bacteria might also metabolize dimethylamine as a substrate to other substances.
+So the results show that expressed TMADH could metabolize TMA into DMA successfully. Also it can be seen that the mutant has higher activity than  normal one, which is in line with the model work.
+We also hope that our results will be helpful to future teams and provide them with some information for reference.
+===References===
+[1] Jang M H, Basran J, Scrutton N S, et al. The reaction of trimethylamine dehydrogenase with trimethylamine[J]. Journal of Biological Chemistry, 1999, 274(19): 13147-13154.
+Because trimethylamine dehydrogenase (TMADH) and dimethylamine dehydrogenase (DMADH) exist as dimers
+[2] Scrutton N S, Sutcliffe M J. Trimethylamine dehydrogenase and electron transferring flavoprotein[J]. Enzyme-Catalyzed Electron and Radical Transfer, 2000: 145-181.