Composite

Part:BBa_K1866007

Designed by: Stefan Oehler   Group: iGEM15_IIT_Delhi   (2015-09-18)

NosZ+SuperYellowFluorescenceProtein (in pSB1C3)

This part consists of NosZ gene and a sequence encoding for Super Yellow Fluorescence Protein downstream to it.

NosZ gene product catalyses the conversion of Nitrous Oxide (N2O) to molecular Nitrogen gas in a process called denitrification.

This part can be cloned under the control of a promoter of choice and its expression can be readily analyzed by checking for the expression of the yellow fluorescence protein.

Gene construction & cloning:

The general cloning strategy for NosZ+SYFP consists of the following steps.

  • 1. The biobrick BBa_K1356006 (NosZ), previously submitted by BYU iGEM 2014 team, was obtained from iGEM repository.
  • 2. Another biobrick consisting of the Super yellow Fluorescence Protein BBa_K864101 (RBS+SYFP) submitted by Uppsala iGEM 2012 team was also procured.
  • 3. (RBS+SYFP) was doubly digested by XbaI and PstI enzymes to yield XbaI-(RBS+SYFP)-PstI construct.
  • 4. (NosZ) was doubly digested by EcoRI and SpeI to yield EcoRI-(NosZ)-SpeI construct.
  • 5. A linearized vector was prepared by double-digestion with EcoRI and PstI.
  • 6. The 3A assembly was performed to clone the desired construct as shown below into the linearized backbone.

    EcoRI-XbaI-(NosZ)-(RBS+SYFP)-SpeI-PstI

    Confirmation of the clone:

    We performed a double digestion by EcoRI and PstI to release the cloned fragment from the recombinant vector. The digestion products were resolved on an Agarose Gel by Electrophoresis and the size of the fragment confirmed the correct clone.


    Figure1: Clone confirmation

    Clone_confirmation_IIT_D_2015.jpg


    Sequence and Features


    Assembly Compatibility:
    • 10
      INCOMPATIBLE WITH RFC[10]
      Illegal PstI site found at 1845
    • 12
      INCOMPATIBLE WITH RFC[12]
      Illegal PstI site found at 1845
    • 21
      INCOMPATIBLE WITH RFC[21]
      Illegal BglII site found at 1517
    • 23
      INCOMPATIBLE WITH RFC[23]
      Illegal PstI site found at 1845
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal PstI site found at 1845
      Illegal NgoMIV site found at 908
      Illegal NgoMIV site found at 1072
      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


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