Device

Part:BBa_K323088

Designed by: Tina Lebar   Group: iGEM10_Slovenia   (2010-10-20)
Revision as of 14:02, 26 October 2010 by TinaL (Talk | contribs)

In vivo testing device for protein-DNA binding: part 1 (DTER_pSYN_BbsI)

This part contains a double terminator (Part:BBa_B0015), and a synthetic promoter (pSYN), designed to contain a BbsI restriction site (operator clone in site). The terminator in front of the promoter serves the purpose of stopping the translation under promoters present in the vector. Combined with Part:BBa_K323089 (containing the corresponding DNA binding protein), this part forms a universal device for in vivo testing of protein-DNA binding.

Figure: BbsI restriction site. The BbsI restriction endonuclease cuts the DNA outside of its recognition site. Primers for the annealing of the operator sequences were designed to fit into the clone in site, that is GACA sequence at the 5` end of the forward primer and AATA sequence at the 3` end of the reverse primer.
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 added to the media, 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
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None