Part:BBa_K1742000

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. [http://www.google.com TEXT]

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.

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]