Coding
AsLOV2pep

Part:BBa_K1742000

Designed by: Ruben Escriba Piera   Group: iGEM15_Valencia_UPV   (2015-09-03)

Avena sativa LOV2 domain

One of the main parts of our revolutionary AladDNA circuit is composed by the second LOV (light-oxigen-voltage) domain of Avena sativa phototropin. LOV domains are small domains (125 amino acids) that bind to flavin mononucleotide (FMN) cofactor. Photoactivable LOV domains have been used in several designs to control cell signaling with high spatial and temporal resolution in bacteria, yeast and mammalian cells [1][2]. Blue light induce conformational alterations in the LOV2 domain and provoke a structural unwinding of C-terminal alpha helix (referred as Jα). We have used the “tunable light inducible dimerization tag” (TULIP) approach [3] where an epitope tag for binding to an engineered PDZ domain (ePDZ) is fused to the Jα helix.


USAGE AND BIOLOGY

For light-induced expression of target genes, there are two part modules that need to be mentioned: One consists of the LOV2 domain that is fused to a DNA-binding domain (BD). This part binds to a specific DNA sequence, an operator sequence upstream to a minimal promoter and the target gene. The second part includes the ePDZ domain (BBa_K1470005) that is fused to the VP16 domain, an activation domain that recruits the transcriptional machinery to the gene of interest. The key process of LOV2 blue light-controlled switch is the interaction between the ePDZ domain with the peptide epitope tagged in the Jα of the LOV2 domain. A great advantage of this system is that the affinity of the interaction can be widely modulated. In the dark state, the Jα helix is not exposed to its ligand, preventing the recruitment of the ePDZ-VP16 domain, and the gene of interest is not transcribed. However, upon illumination with blue light, the Jα unwinds from the LOV2 domain becoming more accessible and enabling the second part, ePDZ fused to VP16, to bind to the Jα peptide. Thus, VP16 recruits RNA polymerase and the gene of interest is transcribed.

800px-ToggleAsLOV.png

Figure 1. Representative scheme of the LOV2 blue light-controlled gene expression system


In order to see if our blue light-dependant switch was functional in plants, the gene of interest assembled (GoldenBraid assembly) with the chimeric promoter (composed by the DBD operator ‘OpLacIBD’, a minimal promoter ‘miniP35S’ and a 5’-UTR region from TMV) was the Luciferase. In order to have a control for the Luciferase assay, we needed another construct already available in the GoldenBraid collection. It included the Renilla and P19 genes.

703px-BBa_K1742000_AsLOV_Luc_AssayFIXED.png

Figure 2. Luciferase assay showing the luciferase/renilla ratios.

References
1. Zhang K., Cui., B (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology. 33: 92-100
2. Levskaya A., Weiner OD., Lim WA. Voigt CA (2009). Spatiotemporal control of cell signaling using a light-switchable protein interaction. Nature 461: 997-1001
3. Strickland D, Lin Y, Wagner E, Hope CM, Zayner J, Antoniou C, Sosnick TR, Weiss EL, Glotzer M (2012). TULIPs: tunable, light-controlled interacting protein tags for cell biology. Nat Methods 9:379-384

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]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
None