Part:BBa_K4238003

Act1p+NLS-VP16-EL222+CYC1t
This part is responsible for the acceptance of blue light in yeast cells. EL222 dimerizes under blue light and binds specifically to a particular DNA sequence (C120). NUS Singapore 2022 created Basic Parts by adding the transcription inducers VP16 and NLS to EL222 to promote specific expressions under blue light. We have implemented this part (BBa_K3570021) with the Constitutive promoter ACT1 and the CYC1 terminator in yeast.

Biology

EL222, which consists of a light-oxygen-voltage-sensing (LOV) domain and a DNA binding domain, helix-turn-helix (HTH) domain, is connected to an activation domain (AD), VP16. In the dark, this complex does not bind to DNA because the LOV domain masks the HTH domain. When blue light (450 nm) hits them, the structure of the LOV domain changes, freeing the HTH domain, which binds to each other to form a homodimer that binds to the C120 region in the DNA. The VP16 domain then promotes the expression of the downstream gene [1][2].

Wet lab result

Plasmid:

with using BBa_K4238004 as a reporter, we tested how EL222 activates C120-trcuCYC1 promoter under the blue light.

Reference

[1] Motta-Mena, L. B., Reade, A., Mallory, M. J., Glantz, S., Weiner, O. D., Lynch, K. W., & Gardner, K. H. (2014). An optogenetic gene expression system with rapid activation and deactivation kinetics. Nature chemical biology, 10(3), 196-202. doi: 10.1038/nchembio.1430 [2] "Team:NUS Singapore - 2021.igem.org." https://2021.igem.org/Team:NUS_Singapore (accessed Oct. 09, 2022)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 801
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 801
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 801
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 801
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 801
  
• 1000
  COMPATIBLE WITH RFC[1000]