Latest revision as of 23:55, 27 October 2020

VVD linked to YC155 (YFP Cter split) with promoter 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 Fluorescent 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 C terminal of the YFP split. The upstream part of this composite is T7 promoter (BBa_I712074) which is strong promoter from T7 bacteriophage.

VVD - C terminal Split YFP

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.

Fig. 2: System VVD - split YFP

VVD - C terminal Split YFP construction

This composite part is composed of the T7 promoter (BBa_I712074), RBS (BBa_J61100) and the composite part VVD links with FOS linker to the C terminal of the YFP split (BBa_K1616021). This part has been created in order to work with BBa_K1616002.

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

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 C terminal of YFP split(1).

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.

Vvd improving

SMS_Shenzhen improved this part in 2020

SMS_Shenzhen combines this part with Spacer between Photoswitches, lac operator, GS linker 1, GS linker 2, and Vvd, forming the J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883. This lead to active T7RNAP.

Experiments & Results

1、Photoswitches efficiency test

Experimental setup

- In this experiment, we transform [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] and plasmid contains GFP regulated by T7 promoter simultaneously into DH5α. We culture the strain overnight to get bacteria culture.
- Bacteria cultures overnight are inoculated 1:200 in fresh LB medium for two times each. One is exposed to blue light, and the other one is covered by tin foil, to protect it from light.
- Take 1mL cell culture for each measurement.
- harvest the cells by centrifuge. Discard the supernatant and add 1mL PBS. Blow the bacteria to dissolve.
- Repeat step 2 for three times, to exclude the deviations influenced by the medium.
- Take 100μL cell culture and mix it with 900μL PBS. Sufficiently mix. - Transferred 200μL of it to a 96 well plate.
- Measure absorbance under OD 600 nm and the fluorescent intensity by a microplate reader. The excitation light is 485nm, and emission light is 535nm.

Results

Test photoswitch efficiency. Here is the result of the photoswitches efficiency test, which is what we have mentioned before in the experiment part. After we have harvested the cells, they are applied the fluorescence intensity measurement.
We divide fluorescence intensity by OD 600nm and get a relative GFP expression condition. The values are applied to view the photoswitches efficiencies by ratio. To convey the result more directly, we illustrate the following column diagram. A truncation is made by us to put all data in one graph.

Measurement on photoswitches' efficiency

Only effective photoswitches are presented in the graph. In these groups, blue bars (Light) are higher than the black bars (Dark).
This means GFP is expressed in a larger amount in Light groups. This indicates a successful construction of photoswitches.
From the Figure, we find pMag-nMag cannot achieve light regulation. Therefore, we choose [[Part:BBa_K3628023|J23106-RBS-T7 RNA polymerase N 1~179-pMagFast2-RBS-nMagHigh1-T7 RNA polymerase C 180~883] and [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] to regulate levodopa yielding in the next experiment.