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.
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
- 12Illegal NheI site found at 4441
- 21Illegal BamHI site found at 4381
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 4216