Part:BBa_K323039
Protein assembly DNA program
Brief description
This unique BioBrick part carries a DNA program sequence with 7 various zinc finger binding sites. In 5' to 3' direction following zinc finger binding elements are embedded: Tyr456_O, Jazz_O, Blues_O, Zif268_O, PBSII_O, ZNF_HIVC_O, Gli1_O. O standing for operator. It can serve to assemble various functional protein domains and bring them in close proximity, therefore enabling the selected system to function better (faster and/or with desired outcome). [http://2010.igem.org/Team:Slovenia Team Slovenia] used this part to reconstitute FRET and test binding of zinc finger domains using surface plasmon resonance ([http://2010.igem.org/Team:Slovenia/PROJECT/proof/studies/spr SPR]) and electrophoretic mobility shift assay ([http://2010.igem.org/Team:Slovenia/PROJECT/proof/studies/emsa EMSA]).
Usage notes
FRET or Forster resonance energy transfer is a non-radiative energy transfer between donor and acceptor molecule. Donor molecule is excited by a particular laser wavelength and transfers part of its energy to its neighbouring fluorescent protein. One of the most characterized FRET pairs are mCerulean (cyan fluorescent protein, CFP) and mCitrine (yellow fluorescent protein, YFP). iGEM Team Slovenia designed a FRET device of completely new kind which was based on binding four chimeric proteins to their specific binding sites on a predesigned DNA program molecule (this part). These can be achieved by using chimeric proteins with DNA binding domain (zinc fingers) and split fluorescent protein half attached to it via a linker sequence.
Video showing this part at work
How this device works is explained in the following video: [http://www.youtube.com/watch?v=MWFV3LKDVyc protein assembly on a DNA program].
Scheme
DNA directs binding of selected functional domains with DNA binding domains. To observe FRET and in order to quantify it we had to fixate cells using paraformaldehyde and bleach cells with 514nm laser light. Reconstitution of split GFPs setting up this FRET device lasts for one night after tranfection into HEK293 cells. It is predicted this can be observed even sooner.
FRET system BioBricks - binding chimeric proteins
Selection of a spacer
We selected 2bp spacer between every bindig site. This was based upon the literature since such reconstitution experiments are not a novelty. What is novel and represents state of the art is simultaneous reconstitution of two fluorescent proteins on a same stretch of DNA.
Applications
Most profound application one can imagine out of such systems is ordering of biosynthetic enzymes to a DNA program and/or implementing simple logical cicuits based on FRET. Directed and most importantly predictive binding to such sequence can: reduce sideway product formation, enhance metabolic flow, process information via phosphorylase domains et. etc. Especially in the field of industrial biotechnology possibilities are limitless.
Future potentials
BioBricks involved in this DNA program based FRET system are composed of a DNA binding domain and a split GFP half which can both be used separately. Meaning one can attach zinc fingers to any other functional domain, not GFP, say an enzyme. At the same time it is possible to use split GFP for say protein-protein interaction studies or DNA origami studies to determine spatial orientation of a protein or a stretch of DNA molecule.
Reference
[1] Hu C. D., Kerpolla K. T. 2003. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nature Biotechnology. 21: 539-545
[2] Segal et al. 2005. DNA Sequence-Enabled Reassembly of the green fluorescent protein. Journal of the Americal Chemical Society. 31: 10782-10783
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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