In vivo testing device for protein-DNA binding: part 1 (DTER_pSYN_BbsI)
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. this part is 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;
2. lacZ reporter gene (Part:BBa_I732019), which expression is controlled by pSYN;
3. DNA binding protein (cut with XbaI/NotI) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI (Part:BBa_K323089).
The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower 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
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC