Device

Part:BBa_K323088

Designed by: Tina Lebar   Group: iGEM10_Slovenia   (2010-10-20)
Revision as of 21:48, 25 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. 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 TTAT sequence at the 3` end of the forward primer and CTGT sequence at the 5` end of the reverse primer). The terminator serves the purpose of stopping the translation under promoters present in the vector.

Combined with Part:BBa_K323089 (containing the relevant DNA binding protein), this part forms a universal testing device for DNA binding proteins.

Binding 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 a promotor, containing 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.

Figure: Universal testing device for DNA binding proteins. A) Expression of the beta-galactosidase without arabinose added to the media: Expression of the DNA binding protein is regulated by the pBAD promoter - if no arabinose to induce the promoter is added, the DNA binding protein cannot be transcribed and therefore cannot bind to its operator sequence, inserted into the pSYN promoter. Beta-galactosidase is thus transcribed and the measured beta-galactosidase activity should be high. B) Expression of the beta-galactosidase with 1% arabinose added to the media: if arabinose is 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.

The 2010 iGEM team Slovenia have tested seven DNA binding proteins with this device. Results can be seen under the 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
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