Difference between revisions of "Part:BBa K4169015"

m (→‎Engineering Success)
Line 38: Line 38:
  
  
−
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.
+
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 5×10-5mol/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>
 
<html>

Revision as of 19:28, 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.


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.

Sequence and Features

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]