Difference between revisions of "Part:BBa K896004"
 
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Cloning of NosZ+NorCB gene:
 
Cloning of NosZ+NorCB gene:
  
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NorCB is known as nitric oxide reductase CB complex, and it is responsible for reducing nitric oxide into nitrous oxide. On the other hand, NosZ stands for the structure gene of nitrous-oxide reductase, which conducts the ultimate process of denitrification; that is, reducing nitrous oxides into nitrogen. In addition, both NorCB and NosZ are originated from Pseudomonas aeruginosa PAO1.
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NosZ-NorCB
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NorCB is composed of two sub-unit – nitric oxide reductase subunit C and B. In fact, these two subunits are adjacent genes. And thus we cloned them together to produce a functional reductase.
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NosZ is the structure gene of nitrous reductase. Furthermore, it performs the last procedure of bio-denitrification in our model. Obviously, the reason we cloned NorCB and NosZ on the same plasmid is to connect the continuous denitrifying procedures together.
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In addition, both NorCB and NosZ are originated from'' Pseudomonas aeruginosa PAO1''.
 
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Latest revision as of 17:56, 25 September 2012

NosZ-NorCB

Cloning of NosZ+NorCB gene:

NosZ-NorCB

NorCB is composed of two sub-unit – nitric oxide reductase subunit C and B. In fact, these two subunits are adjacent genes. And thus we cloned them together to produce a functional reductase.

NosZ is the structure gene of nitrous reductase. Furthermore, it performs the last procedure of bio-denitrification in our model. Obviously, the reason we cloned NorCB and NosZ on the same plasmid is to connect the continuous denitrifying procedures together.

In addition, both NorCB and NosZ are originated from Pseudomonas aeruginosa PAO1.


We clone NosZ and NorCB and ligate them together. However, since there are PstI enzyme cutting site inside the NorCB, so we clone a new pSB1C3 backbone which contain EcoRI.XbaI.SpeI.SbfI enzyme cutting site.

NosZ+NorCB cloning.jpg

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1845
    Illegal PstI site found at 2489
    Illegal PstI site found at 3605
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1845
    Illegal PstI site found at 2489
    Illegal PstI site found at 3605
    Illegal NotI site found at 3764
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1517
    Illegal BamHI site found at 2658
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1845
    Illegal PstI site found at 2489
    Illegal PstI site found at 3605
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1845
    Illegal PstI site found at 2489
    Illegal PstI site found at 3605
    Illegal NgoMIV site found at 908
    Illegal NgoMIV site found at 1072
    Illegal NgoMIV site found at 2684
    Illegal NgoMIV site found at 2726
    Illegal NgoMIV site found at 3628
    Illegal AgeI site found at 282
    Illegal AgeI site found at 1381
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1638
    Illegal BsaI.rc site found at 667
    Illegal BsaI.rc site found at 1312
    Illegal BsaI.rc site found at 1940