Part:BBa_K1616002

VVD linked to YN155 (YFP Nter split) with double terminator T7

The VVD receptor

Fig. 1: Models of signaling in LOV domains VVD

Vivid (VVD) is the smallest known Light–oxygen–voltage (LOV) domain protein and photo-inducible dimer. Isolated from Neurospora crassa, VVD forms a homodimer in response to a blue-light stimulus. The LOV domain, present in VVD, is a small blue-light sensing domain found in prokaryotes, fungi and plants. After blue-light activation, a covalent bond is formed between the co-factor Flavin mononucleotide (FMN) and one of the cysteine residue. This bond leads to a conformational change inducing functions by dissociating the C-terminal a-helix (Ja) and the LOV-core. In VVD, this undock triggers homodimerization (Bilwes, Dunlap, & Crane, 2007; Müller & Weber, 2013).

Contrary to other photoreceptors, VVD is a small protein with 150 amino-acids facilitating accurate molecular design and avoiding steric issues (BBa_K1616014). Moreover, it is a homo-dimer when most of photo-inducible dimers are heterodimers. In addition, the use of VVD is easy; and doesn’t need any addition of co-factors: VVD works with Flavin adenine dinucleotide (FAD) which is already abundant in eukaryote and prokaryote cells (Müller & Weber, 2013; Nihongaki, Suzuki, Kawano, & Sato, 2014).

Split Protein

Yellow Frluorescent Protein

A split protein is a protein whose sequence has been divided into two (or more) different parts. Often used to study protein-protein interactions, the protein can not perform its function until the parts are put back together. The yellow-fluorescent (YFP), the yellow-fluorescent protein, will only express fluorescence when its two parts will be reunited.

In normal condition, the production of a protein in response to a stimulus can easily reach several hours due to the many steps required for the protein synthesis. By using split-proteins, we are taking advantage of the absence of fluorescence when the two parts are apart. Indeed, the two parts of our split-YFP, when remaining separated, can be produced without being effective. Therefore, the overall process is far less time-consuming. However, to implement a light control on the fluorescence activation, a genetic construction gathering the VVD photoreceptor and our split-YFP has to be engineered.

Biomolecular fluorescence complementation

The new alternative approach for the visualization of protein interactiosn has been developed; the biomolecular fluorescence complementation (BiFC) techniques based on the complementation between fragments of fluorescent proteins; fragments of the yellow fluorescent protein (YFP) brought together by the association of two interaction partners fused to the fragments. They noticed that the spectral characteristics of BiFC of YFP were virtually identical to those of intact YFP.(Chang-Deng Hu, 2003)

VVD - Split YFP

The idea is to induce bacteria fluorescence through light signals. For this, we have added to each part of the YFP-split the VVD homodimer, so we have a system triggering by light inducing fluorescence. The part is coding for the homodimer VVD links by an integration of specific sequence to the N terminal of the YFP split. The downstream part of this composite is double T7 terminator (BBa_B0015).

VVD - N terminal Split YFP

So, this part works with BBa_K1616001. In absence of blue-light, the conformation of the VVD photoreceptor will prevent the formation of the complete fluorescent protein while in presence of the light signal the YFP protein will be reconstituted leading to the fast expression of a yellow fluorescence in our bacteria.

Fig. 2: System VVD - split YFP

VVD - N terminal Split YFP construction

This composite part is composed of RBS (BBa_J61100), the composite part VVD links with JUN linker to the N terminal of the YFP split (BBa_K1616020) and the T7 double terminator(BBa_B0015). This part has been created in order to work with BBa_K1616001.

Fig. 3: The complete part for the VVD - YFP split system

VVD - C terminal Split YFP construction

Electrophoresis after EcoRI digestion to check the Biobrick

Expected results

Results

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]

Design Notes

All BioBrick parts used for assembling the composite part are compatible with the RFC10 Biobrick standard. The original sequence of VVD had 2 illegal sites PstI; that have been removed.

Source

This part have been created thank to gblock, our team have assembled the sequence of photoreceptor VVD (without illegal site), a linker(1) and then the N terminal of YFP split(1). The downstream part of this composite part is double T7 terminator (BBa_B0015) and the upstream is the RBS (BBa_J61100).

References

(1) Tom Kerppola, Ph. D, investigator at the Howard Hughes Medical Institute as well as Professor in the University of Michigan

Bilwes, A. M., Dunlap, J. C., & Crane, B. R. (2007). Conformational Switching in the Fungal Light Sensor Vivid, 36(May), 1054–1058.
Chang-Deng Hu, T. K. K. (2003). Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nature Biotechnology, 21(5), 539–545.
Hu CD, Chinenov Y, Kerppola TK. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell. 2002;9(4):789–98.