Part:BBa_K678061

pJEJAM13

pJEJAM13 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 , GFP with the peroxisomal targeting sequence (PTS1) at 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 pJEJAM1, the figure is not drawn to scale.

Green fluorescence can be observed in the hyphae and in clear spots (Figure 3). This indicates that GFP is targeted to the peroxisomes, but further investigations are required to confirm this. In figure 4 and figure 5 it be can see that the integration of the linearized pJEJAM13 must have impaired a gene or pathway of the transformant because its morphology and growth differed significantly from the [http://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Wild%20type wild-type].

DTU-Denmark-2 2011 Figure 2: DIC image of Aspergillus nidulans with pJEJAM13.

DTU-Denmark-2 2011 Figure 3: 'Fluorescence image of Aspergillus nidulans with pJEJAM13. Green fluorescence can be observed in the hyphae and in clear spots.

DTU-Denmark-2 2011 Figure 4: Aspergillus nidulans Front

DTU-Denmark-2 2011 Figure 5: Aspergillus nidulans Back

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 3777
  Illegal XbaI site found at 5333
  Illegal XbaI site found at 8077
  Illegal PstI site found at 140
  Illegal PstI site found at 4618
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 910
  Illegal PstI site found at 140
  Illegal PstI site found at 4618
  Illegal NotI site found at 5290
  Illegal NotI site found at 8116
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 144
  Illegal BamHI site found at 3058
  Illegal BamHI site found at 4356
  Illegal XhoI site found at 700
  Illegal XhoI site found at 1082
  Illegal XhoI site found at 4216
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3777
  Illegal XbaI site found at 5333
  Illegal XbaI site found at 8077
  Illegal PstI site found at 140
  Illegal PstI site found at 4618
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3777
  Illegal XbaI site found at 5333
  Illegal XbaI site found at 8077
  Illegal PstI site found at 140
  Illegal PstI site found at 4618
  Illegal NgoMIV site found at 3694
  Illegal AgeI site found at 1289
  Illegal AgeI site found at 1437
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4409
  Illegal BsaI.rc site found at 1116
  Illegal BsaI.rc site found at 3408
  Illegal SapI site found at 322