Part:BBa_K323089

In vivo testing device for protein-DNA binding: part 2 (lacZ_DTER_pBAD_BsaI_DTER)

This part is composed from a lacZ reporter gene (Part:BBa_I732019), a double terminator (Part:BBa_B0015) and Part:BBa_K323110, which our team designed previously. Combined with Part:BBa_K323088 (containing the corresponding binding sequence), this part forms a device for in vivo testing of protein-DNA binding. Part:BBa_K323110 includes a BsaI clone in site inbetween a promoter and a terminator, intended for insertion of any chosen DNA binding protein gene.

Figure: BsaI restriction site. The BsaI restriction endonuclease cuts the DNA outside of its recognition site. The clone in site was designed in such a way, that any BioBrick gene, cut with XbaI and NotI enzymes could be ligated into the vector, cut with the BsaI enzyme.

Figure: Universal testing device for DNA binding proteins. A) Expression of beta-galactosidase with no arabinose present: Expression of the DNA binding protein is regulated by the pBAD promoter. With no arabinose to induce the promoter, the DNA binding protein cannot be transcribed and therefore cannot bind to its operator sequence, inserted into the pSYN promoter. The lacZ gene is thus transcribed and the measured beta-galactosidase activity high. B) Expression of beta-galactosidase with arabinose present: With arabinose added to the media, the pBAD promoter is induced, the DNA binding protein is transcribed and bound to its operator sequence in the pSYN promoter. Therefore the promoter is inactive and the lacZ gene cannot be transcribed, which results in a low beta-galactosidase activity.

Binding of the DNA binding proteins can be tested with the beta-galactosidase assay (for detailed description of the method see the 2010 iGEM team Slovenia wiki). The activity of a beta-galactosidase enzyme, expressed under the promotor with a binding site for a DNA binding protein, is measured. By this principle the binding strength of a chosen DNA binding protein to its binding sequence can be determined - the stronger the binding, the lower the expression of beta-galactosidase under the pSYN promoter.

The 2010 iGEM team Slovenia have tested seven DNA binding proteins (zinc fingers HivC, Gli1, Zif268, Jazz, Blues, PBSII and the TAL transcription factor) with this device. For results of the experiment see tab "Experience".

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 4441
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 4381
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 4216
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4496
  Illegal BsaI.rc site found at 4472
  Illegal SapI site found at 4198