Part:BBa_K896004

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.

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