Assembly of the 2 parts of VVD and split YFP
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. Then, a split protein is a protein whose sequence has been divided into two (or more) different parts. The yellow-fluorescent (YFP) protein will only express fluorescence when its two parts will be reunited. 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). 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.
The sequence of VVD had 2 illegal sites PstI; that have been removed. Also, the N terminal of YFP had 1 illegal site PstI
VVD linked to YC155 (YFP Cter split) with promoter T7
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 C terminal of YFP split(1). (1) Tom Kerppola, Ph. D, investigator at the Howard Hughes Medical Institute as well as Professor in the University of Michigan
Vivid (VVD) is the smallest known Light???oxygen???voltage (LOV) domain protein and photo-inducible dimer. Isolated from Neurospora crassa, VVD forms a homo-dimer in response to a blue-light stimulus. Then, a split protein is a protein whose sequence has been divided into two (or more) different parts. The yellow-fluorescent (YFP) protein will only express fluorescence when its two parts will be reunited. The Part is coding for the homodimer VVD link by an integration of specific sequence to the C terminal of the YFP split. So, this part works with BBa_K1616002. 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.
The sequence of VVD had 2 illegal sites PstI; that have been removed.
VVD linked to YN155 (YFP Nter split) with double terminator T7
Assembly the sequence of photoreceptor VVD (without illegal site), a linker(1) and then the N terminal of YFP split(1)
(1) Tom Kerppola, Ph. D, investigator at the Howard Hughes Medical Institute as well as Professor in the University of Michigan 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.
Double terminator consisting of BBa_B0010 and BBa_B0012
T7 promoter (strong promoter from T7 bacteriophage)
T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
Assembly of 3 parts: BBa_K1616014, BBa_K1616015 and BBa_K1616016 without scar
See BBa_K1616001. This part have been created in order to have the three parts without scars (VVD,linker and YFP Nterminal).
Assembly of 3 parts: BBa_K1616014, BBa_K1616018 and BBa_K1616017 without scars
See BBa_K1616002. This part have been created in order to have the three parts without scars (VVD,linker and YFP Nterminal).
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
VVD was isolated from Neurospora crassa.
Among other photoreceptors, Vivid (VVD) is the smallest known Light???oxygen???voltage (LOV) domain protein and photo-inducible dimer. Isolated from Neurospora crassa, VVD forms a homo-dimer 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; M??ller & Weber, 2013). Contrary to other photoreceptors, VVD is a small protein with 150 amino-acids facilitating accurate molecular design and avoiding steric issues. 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 (M??ller & Weber, 2013; Nihongaki, Suzuki, Kawano, & Sato, 2014).
We found illegal sites (PstI) into VVD sequence, those one have been removed.
Linker used in association with VVD (BBa_K1616014)
This part is a sequence coding for 17 aa useful to link two proteins
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. For instance, 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. 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)
Linker used in association with YFP Nterminal split and VVD.
This part is a sequence coding for 5 aa useful to link VVD and YFP N terminal split protein.
Conversion of the iGEM parts registry to SBOL2.1