Part:BBa_K678060

pJEJAM12

pJEJAM12 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 (NHEJ). The plasmid consists the strong constitutive gpdA promoter, green fluorescent protein (GFP), 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 pJEJAM12, the figure is not drawn to scale.

When pJEJAM12 was integrated into A. nidulans expression of the gene encoding GFP, resulted in an even distribution of GFP in the hyphae (Figure 3). When comparing figure 2 and figure 3 it can be seem that GFP was not expressed in all hyphae, this could be as a result of the integration by NHEJ as we in figure 3 and figure 4 can see that the integration must have impaired a gene or pathway of the transformant, a picture of the wild-type strain can be found [http://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/fungi#Wild%20type here].

DTU-Denmark-2 2011 Figure 2: DIC image of Aspergillus nidulans with pJEJAM12.

DTU-Denmark-2 2011 Figure 3: Fluorescence image of Aspergillus nidulans with pJEJAM12.

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 3768
  Illegal XbaI site found at 5324
  Illegal XbaI site found at 8068
  Illegal PstI site found at 140
  Illegal PstI site found at 4609
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 910
  Illegal PstI site found at 140
  Illegal PstI site found at 4609
  Illegal NotI site found at 5281
  Illegal NotI site found at 8107
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 144
  Illegal BamHI site found at 3049
  Illegal BamHI site found at 4347
  Illegal XhoI site found at 700
  Illegal XhoI site found at 1082
  Illegal XhoI site found at 4207
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3768
  Illegal XbaI site found at 5324
  Illegal XbaI site found at 8068
  Illegal PstI site found at 140
  Illegal PstI site found at 4609
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3768
  Illegal XbaI site found at 5324
  Illegal XbaI site found at 8068
  Illegal PstI site found at 140
  Illegal PstI site found at 4609
  Illegal NgoMIV site found at 3685
  Illegal AgeI site found at 1289
  Illegal AgeI site found at 1437
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4400
  Illegal BsaI.rc site found at 1116
  Illegal BsaI.rc site found at 3399
  Illegal SapI site found at 322