Part:BBa_K1936001

Tom5-eGFP-LOV2

ALERT !!!Under construction !!

Usage

While LOV2 domain was previously described for optogenetical control of gene expression (BBa_K1742000) and as reporter gene (BBa_K660003), we took a new approach. The described fusion protein Tom5-eGFP-LOV2 is designed to recruit proteins of choice to the outer mitochondrial membrane (OMM) in engineered eukaryotic cells.

Tom5 is a part of the eukaryotic translocase of outer membrane protein complex and has a single membrane anchor unit that integrates into OMM. [1] We fused it to the N-terminal end of LOV2 domain from A.sativa. Upon exposure to blue light (λ=473nm) the LOV2 peptide undergoes a conformational change and exposes its C-terminal J-alpha-helix which is then able to bind a specifically engineered PDZ domain (BBa_K1470005). [2] The fused enhanced green fluorenscence protein (eGFP) allows the tracking of cellular localization by microscopy.

Our blue light dependent system can be used to recruit any protein of interest to the outer mitochondrial membrane by simply fusing it to an ePDZ domain.

Fig.1 Activation of the blue light dependent OMM recruitment system.

For our project we utilized the light dependent OMM recruitment as a potential method for cancer treatment. As shown in Fig.1 it recruits BAX protein which is fused to the ePDZ domain (see BBa_K1936000). Once BAX is localized at the mitochondrial membrane it leads to formation of apoptotic channels. Through those channels cytochrome c can be released from the mitochondrial intermembrane space to the cytosol which triggers apoptosome formation. This allows us to kill abnormal cells with attainment of a very high level of spatiotemporal specificity compared to traditional cancer therapies (http://2016.igem.org/Team:Duesseldorf).

Design

We choosed Tom5 from Saccharomyces cerevisiae as OMM anchor domain for our construct. It has been shown to be structurally very similar to its mammalian homologues. [3]

The LOV domain originated from Avena sativa. The T406A,T407A double mutation has been shown to stabilize the N-terminal A′α helix of AsLOV2 which reduces the dark activity of J&#945 helix while also increasing its docking affinity to ePDZ. Due to the C-terminal position of the J&#945 helix Tom5 and eGFP were fused to the N-terminal side of LOV2 peptide. [2]

References

[1] https://www.ncbi.nlm.nih.gov/pubmed/9217162

[2] 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. 2012 Mar 4;9(4):379-84. doi: 10.1038/nmeth.1904.

[3] Kato H, Mihara K (2008) Identification of Tom5 and Tom6 in the preprotein translocase complex of human mitochondrial outer membrane. Biochem Biophys Res Commun. 2008 May 9;369(3):958-63. doi: 10.1016/j.bbrc.2008.02.150. Epub 2008 Mar 10.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 33
  Illegal BamHI site found at 712
  Illegal XhoI site found at 515
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1226