Part:BBa_K678063

pJEJAM15

pJEJAM15 is a plasmid that after digestion with NotI is intended to be transformed into Aspergillus nidulans and integrated into the genome by non-homologous end joining. The plasmid consists the strong constitutive gpdA promoter , mRFP with a nucleosomal targeting sequence (NLS) fused to the C-terminus, the trpC terminator, a pyrG marker cassette for selection, an ori for propagation in E. coli, and an ampR resistance cassette (Figure 1).

DTU-Denmark-2 2011 Figure 1: Plasmid map of pJEJAM15, the figure is not drawn to scale.

Red fluorescence can be observed in clear spots in Figure 3. When comparing Figure 2 and FIgure 3 it can be seem that both spores and hyphae fluoresce. We assume that the fusion of mRFP1 with the NLS results in the targeting to the nucleus. To confirm that the mRFP1 in fact is targeted to the nucleus further experiments would have to be conducted. In figure 4 and figure 5 it be can see that the integration of the linearized pJEJAM15 must have impaired a gene or pathway of the transformant because its morphology and growth differed slightly from the [http://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Wild%20type wild-type].

DTU-Denmark-2 2011 Figure 2: Differential interference contrast image of Aspergillus nidulans with pJEJAM15.

DTU-Denmark-2 2011 Figure 2: Fluorescence image of Aspergillus nidulans pJEJAM15.

DTU-Denmark-2 2011 Figure 2: Aspergillus nidulans Front

DTU-Denmark-2 2011 Figure 2: Aspergillus nidulans Back

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 3747
  Illegal XbaI site found at 5303
  Illegal XbaI site found at 8047
  Illegal PstI site found at 140
  Illegal PstI site found at 2657
  Illegal PstI site found at 4588
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 910
  Illegal PstI site found at 140
  Illegal PstI site found at 2657
  Illegal PstI site found at 4588
  Illegal NotI site found at 5260
  Illegal NotI site found at 8086
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 144
  Illegal BamHI site found at 3028
  Illegal BamHI site found at 4326
  Illegal XhoI site found at 700
  Illegal XhoI site found at 1082
  Illegal XhoI site found at 4186
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3747
  Illegal XbaI site found at 5303
  Illegal XbaI site found at 8047
  Illegal PstI site found at 140
  Illegal PstI site found at 2657
  Illegal PstI site found at 4588
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3747
  Illegal XbaI site found at 5303
  Illegal XbaI site found at 8047
  Illegal PstI site found at 140
  Illegal PstI site found at 2657
  Illegal PstI site found at 4588
  Illegal NgoMIV site found at 3664
  Illegal AgeI site found at 1289
  Illegal AgeI site found at 1437
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4379
  Illegal BsaI.rc site found at 1116
  Illegal BsaI.rc site found at 3378
  Illegal SapI site found at 322