Part:BBa_K819005

Constitutive LuxBrick Generator

a fusion protein consisting of E.coli endogenous SOS system repressor LexA and fungus N.crass photosensor protein VVD with LexA carrying mutation at position 40-42 and VVD carrying mutation N56K, C71V and M135I. This part serves as an ultra sensitive photoreceptor(which can sense light as weak as moonlight) and will induce a light-dependent repression of genes with their promoter containing mutated 408 form of SOS box.

Luminesensor is designed by following the general principle of optogenetics fusion protein design--attaching a physiologically functional domain to a photoreceptor domain.

FIG.1 The general design of optogenetic fusion protein.

In order to circumvent the potential problems of current optogenetic approaches, we concluded that our Luminesensor should be highly sensitive, should incorporate no chromophore that cannot be synthesized by bacteria, and should be modular in structure and function. Based on these standards we can go through the photoreceptor proteins previously used in optogenetic research, examine them one by one, and rule out inappropriate ones one by one to finally reach the most proper light-sensitive photoreceptor domain for our design.We gladly found that among all these three groups of commonly used light-sensitive domains, phototropins possesses the most distinct modular structure. Phototropins have a structurally conserved light sensor domain, termed LOV (light, oxygen and voltage) domain, which is easily discernable and often precedes, within a single reading frame, a sequence of an enzymatically functional domain, connected by the sequence of a linker domain. Despite its structural modularity, the LOV light sensitive domain also seems to possess an outstanding functional modularity, for it was found to combine with various functional domains to perform various cellular functions. Several classes of LOV domain–effector domain combinations have hitherto been described, such as LOV-HKs combination, performing histidine kinase function, LOV GGDEF–EAL combination, predicted to regulate the synthesis and hydrolysis of cyclic di-GMP, and LOV_HTH combination, performing light-induced DNA binding activity. Such versatility already existing in nature strongly suggested that a newly designed artificial fusion protein that fuses the LOV photosensitive domain to a physiologically functional domain to create a non-pre-existing combination would probably work. LOV photosensor domain seems to bear much more virtue than just its modularity. LOV domains have a non-covalently bound flavin (FMN or FAD) chromophore that is absolutely essential for its function. Unlike those choromophres of rhodopsins and phytochormes, flavin is an essential chemical compound deeply involved in the respiratory chain in all forms of life, including bacteria. In another word, in order to let this LOV domain function in bacteria, we only need to incorporate the gene into the cell, and the cell will automatically supply the protein with the chromophore. So far our good phototropin has satisfied two of our requirements: modularity and compatibility. So how about its sensitivity? To date, scientists have, for example, attached Rac1 protein to LOV2 domain from phototropin1 originated from Avena sativa to achieve photoactivatable cell motility, or shuffled the histidine kinase domain of FixL protein, which belongs to a two-component system, to the downstream of the LOV domain of B. subtilis YtvA protein to create a light-regulated histidine kinase, and so on. Among these designs, a particular one that utilizes the photosensor domain of a Vivid (VVD) protein, which originated from Neurospora. Crassa, camptured our attention. The VVD-GAL fusion protein thus designed achieved a rather high light-sensitivity—about 0.04W/m2. This is definitely thrilling news. Moreover, VVD was shown to form a rapidly exchanging homodimer upon blue-light activation, which means, like the Phy-PIF system, the general theme of protein-protein interaction might also be applied to this particular protein. Adding more promise to this VVD protein is its size—the smallest LOV domain containing protein known. This feature makes it easy to engineer and more likely to be stably expressed in bacteria systems.

FIG.2 Illustration of function mechanism of phototropin VVD.

Based on the discussion above, we have finally chosen the smallest LOV protein (or phototropin) VVD protein’s photosensor domain as our photoreceptor domain. Due to its small size, structural and functional modularity, bacteria compatibility, previous experience of being highly sensitive and its photoswitching mechanism, we reasoned that VVD photosensor domain would be an enabling tool in our coming design of a novel optogenetic module.

Our next step is to choose a physiologically functional domain for our new optogenetic module

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 3014
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2012
  Illegal XhoI site found at 2842
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 4401
  Illegal BsaI.rc site found at 1410
  Illegal SapI.rc site found at 4726