Difference between revisions of "Part:BBa K1067003"
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===Characterization===
 
===Characterization===
  
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<img src="https://static.igem.org/mediawiki/2013/e/ed/Dtu_Amo_confirmation.png" width=300px height=200px>
 
[[File:dtu_Amo_confirmation.png|thumb|300px|Ammonia consumption with respect to growth (optical density) for two replicates of the AMO transformant, and untransformed E. coli control.  The rate of consumption of ammonia is much higher in the transformant than in the control. ]]
 
[[File:dtu_Amo_confirmation.png|thumb|300px|Ammonia consumption with respect to growth (optical density) for two replicates of the AMO transformant, and untransformed E. coli control.  The rate of consumption of ammonia is much higher in the transformant than in the control. ]]
  

Revision as of 10:08, 12 October 2013

Ammonium monooxygenase from Nitrosomonas europaea

Ammonia monooxygenase (AMO) from Nitrosomonas europaea converts ammonia to hydroxylamine;

NH3 + O2 + cycAXred + 2 H+ → NH2OH + H2O + cycAXox

The whole operon containing amoA1, amoB1, amoC1 http://ecocyc.org/NEUR228410/NEW-IMAGE?type=MAPTICK&object=2238597/2241670&chromosome=CHROMOSOME-1&bp-range=2238597/2241670 link to Biocyc was extracted by colony PCR on a N. europaea culture.


Usage and Biology

AMO is an integral membrane protein composed of 3 subunits (amoA, amoB, amoC) and is first step in the enzymatic conversion of ammonia to nitrite in the nitrifying bacteria genus. The protein works in close relationship with cycA, cycX and Hydroxylamine oxidoreductase (HAO) in this process. HAO is providing 2 free electrons that are picked up and delivered to AMO by the cytochromes cycA and cycX. The electron from HAO is what makes AMO's work possible.

This part is experienced to slow down growth rates of E.coli substantially; likely because this is a heterologous expressed membrane protein and thereby disrupts E.coli membrane.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 263
    Illegal BamHI site found at 306
    Illegal BamHI site found at 2510
    Illegal BamHI site found at 2822
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2028
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

<img src="Dtu_Amo_confirmation.png" width=300px height=200px>

File:Dtu Amo confirmation.png
Ammonia consumption with respect to growth (optical density) for two replicates of the AMO transformant, and untransformed E. coli control. The rate of consumption of ammonia is much higher in the transformant than in the control.