Difference between revisions of "Part:BBa K4169015"

Revision as of 19:41, 13 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.

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 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 theoretical knowledge.

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 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 V344C mutant and compared it with the unmutated TMADH to quantitatively detect the enzyme activity.
 ===Engineering Success===
@@ Line 46: / Line 48: @@
 <center><b>Figure 2.</b> Concentration Changes of Metabolism Substrate DMA </center>
 <br>
-Results show that expressed TMADH could metabolize TMA into DMA successfully.
+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 theoretical knowledge.
 ===Sequence and Features===