Part:BBa_K323089

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

Device for in vivo testing of protein-DNA binding.

To test binding of DNA binding proteins to a corresponding specific target DNA sequence in vivo we designed a device composed of several parts:

1. DNA binding protein (cut with XbaI/NotI) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI.

2. part Part:BBa_K323088 with a synthetic promoter pSYN in which a DNA binding sequence, particular for each DNA-binding protein to be tested, could be inserted between -35 and -10 sites using BbsI restriction site ;

3. lacZ reporter gene (Part:BBa_I732019), which expression is controlled by pSYN;

The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower beta-galactosidase activity.

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. 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: Device for in vivo testing of protein-DNA binding. 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.

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