Part:BBa_K758005

UAS LacZ, this part expresses LacZ in the presence of GAL4

This part mainly cosists of UAS, LacZ, some terminators, and SV40 polyadenylation signal. SV40 polyadenylation is necessary to exress genes in eukaryote Once GAL4 protein binds UAS sequenses, expression of LacZ is promoted. So, if GAL4 with appropriate promoter exists, this part expresses LacZ in eukaryote.

Experiments and Results

We transfected Drosophila cells with this plasmid, too. We devided the cells into two groups. One group had been co-transfected with pAct5C-GAL4 again, and the other group had been transfected with this plasmid alone.
If BBa_K758005 was assembled accurately, the former group expresses LacZ strongly and the latter expresses weakly.

Generally, to test the activation of LacZ, 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal) is used by mesuring blue color made by the degradation of X-Gal. However, in this experiment, we conducted immunostaining by red color to test the expression of LacZ. Also we use 4', 6-diamidino-2-phenylindole (DAPI) that binds and emit blue light in order to count cells.

As results shown below,some red cells were detected among the cells transfected with pAct5C-GAL4. They prove that LacZ is activated in this group. But the other group without transfection with pAct5C-GAL4 contains no red cells.
We counted cells and calculated ratios of red cells to total cells between these two cases. In consequence, by the transfection of pAct5C-GAL4, 10.1%(42/415) cells showed red color. But, without pAct5C-GAL4, no cells showed red color (0/118 ).

These results show that BBa_K758005 was activated and expression of LacZ were induced in the presence of the GAL4 protein.

Therfore, it can be said that BBa_K758005 was working as expected.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3393
  Illegal PstI site found at 4
  Illegal PstI site found at 247
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3393
  Illegal PstI site found at 4
  Illegal PstI site found at 247
  Illegal NotI site found at 3447
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3393
  Illegal BglII site found at 377
  Illegal BamHI site found at 3614
  Illegal XhoI site found at 3454
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3393
  Illegal PstI site found at 4
  Illegal PstI site found at 247
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3393
  Illegal PstI site found at 4
  Illegal PstI site found at 247
  Illegal AgeI site found at 3519
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3501