Difference between revisions of "Part:BBa K1067003"
(→‎Usage and Biology)
 
(12 intermediate revisions by 2 users not shown)
Line 6: Line 6:
 
'''NH<sub>3</sub> + O<sub>2</sub> + cycAX<sub>red</sub> + 2 H<sup>+</sup> &rarr; NH<sub>2</sub>OH + H<sub>2</sub>O + cycAX<sub>ox</sub>'''  
 
'''NH<sub>3</sub> + O<sub>2</sub> + cycAX<sub>red</sub> + 2 H<sup>+</sup> &rarr; NH<sub>2</sub>OH + H<sub>2</sub>O + cycAX<sub>ox</sub>'''  
  
−
The whole operon containing amoA1, amoB1, amoC1 [[http://biocyc.org/NEUR228410/NEW-IMAGE?type=OPERON-IN-CHROM-BROWSER&object=TUJNO-524 Biocyc]] was extracted by colony PCR on a ''N. europaea'' culture.  
+
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===
 
===Usage and Biology===
−
AMO is and 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 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.  
+
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. AMO converts ammonia into the intermediate hydroxylamine.  This protein works in close relationship with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1067002 cycA], [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1067002 cycX] and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1067004 Hydroxylamine oxidoreductase (HAO)] to complete the nitrification process by converting hydroxylamine to nitrite. HAO is providing 2 free electrons that are picked up and delivered to AMO by the cytochromes cycA and cycX, however another cytochrome from ''E. coli'' can also fill this role and the AMO can function on its own as well.
  
−
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.  
+
===Characterization===
 +
 
 +
https://static.igem.org/mediawiki/2013/2/25/Dtu_amo_small.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.  [https://static.igem.org/mediawiki/2013/e/ed/Dtu_Amo_confirmation.png (larger image)]
 +
 
 +
This part is experienced to slow down growth rates of ''E.coli'' substantially; likely because this is a heterologously expressed membrane protein and thereby disrupts the ''E.coli'' membrane, and because the hydroxylamine product is toxic to ''E. coli''.
  
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1067003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1067003 SequenceAndFeatures</partinfo>
 +
 +
 +
 +
 +
  
  

Latest revision as of 10:27, 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. AMO converts ammonia into the intermediate hydroxylamine. This protein works in close relationship with cycA, cycX and Hydroxylamine oxidoreductase (HAO) to complete the nitrification process by converting hydroxylamine to nitrite. HAO is providing 2 free electrons that are picked up and delivered to AMO by the cytochromes cycA and cycX, however another cytochrome from E. coli can also fill this role and the AMO can function on its own as well.

Characterization

Dtu_amo_small.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. (larger image)

This part is experienced to slow down growth rates of E.coli substantially; likely because this is a heterologously expressed membrane protein and thereby disrupts the E.coli membrane, and because the hydroxylamine product is toxic to E. coli.


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]