This red/far red toggle switch is based on the interaction between proteins expressed by Arabidopsis thaliana, phytochrome B (PhyB) and the phytochrome-interacting factor 6 (PIF6). PhyB is a photosensitive protein that can change its conformation to two different states depending on the light impinging on it. Red light activates the chromophore and allows its binding to PIF6. Far red light causes the disassociation of the proteins inactivating the genetic device.

We also implemented the idea of fusing the transactivation domain of VP16 from herpes simplex virus in order to allow the DNA interaction. And what is more, the N-terminal domain of PIF6 was combined with the different binding domains as Gal4, LacI, LexA and E.

One of the main parts of our revolutionary AladDNA circuit is composed by the second LOV (light-oxigen-voltage) domain of Avena sativa phototropin. LOV domains are small domains (125 amino acids) that bind to flavin mononucleotide (FMN) cofactor. Photoactivable LOV domains have been used in several designs to control cell signaling with high spatial and temporal resolution in bacteria, yeast and mammalian cells [4][3]. Blue light induce conformational alterations in the LOV2 domain and provoke a structural unwinding of C-terminal alpha helix (referred as Jα). We have used the “tunable light inducible dimerization tag” (TULIP) approach [5] where an epitope tag for binding to an engineered Erbin PDZ domain (ePDZ) is fused to the Jα helix.

For light-induced expression of target genes, there are two part modules that need to be mentioned: one consists of the LOV2 domain that is fused to a DNA-binding domain (BD). This part binds to a specific DNA sequence, an operator sequence upstream to a minimal promoter and the target gene.

The second part includes the Erbin PDZ domain (ePDZ) that is fused to the VP16 domain, an activation domain that recruits the transcriptional machinery to the gene of interest.

The key process of LOV2 blue light-controlled switch is the interaction between the ePDZ domain with the peptide epitope tagged in the Jα of the LOV2 domain. A great advantage of this system is that the affinity of the interaction con be widely modulated. In the dark state, the Jα helix is not exposed to its ligand, preventing the recruitment of the ePDZ-VP16 domain, and the gene of interest is not transcribed. However, upon illumination with blue light, the Jα unwinds from the LOV2 domain becoming more accessible and enabling the second part, ePDZ fused to VP16, to bind to the Jα peptide. Thus, VP16 recruits RNA polymerase and the gene of interest is transcribed.

The green fluorescent protein Dronpa derive from a tetrameric parent isolated from a stony coral species (Pectiniidae) that was engineered to a monomeric form using both, rational and random mutagenesis [6]. Dronpa fluorescence is switched off upon illumination with cyan light (500 nm) and switched on again upon illumination with violet light (400 nm). Dronpa is considered as a visualization tool, and it has recently been appreciated as an optical control element. Upon the introduction of a K145N substitution, a homotetrameric complex is formed that monomerizes upon illumination with cyan light. Generating tandem fusions of the original monomeric form DronpaK and the tetrameric mutant DronpaN enables the reversible dissociation of intramolecular dimmers with blue light [7]. This optogenetic method has not been proved as a transgene expression system yet, making it a challenge that only AladDNA can realize.

We have de novo designed and engineered an identical approach for light-directed transcriptional activation of target genes in plants, as mentioned above for AsLOV2-ePDZ.

The first part consists of DronpaK, fused to a DNA-binding domain that binds its operator site nearby the promoter region of a target gene. The second part includes DronpaN fused to the VP16 domain, which acts as a transcriptional activator of a target gene.

Figure 2.

Figure 3.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

The SIP construction was performed by Juarez P (non published data), and kindly provided to us for our experiments. In the construction designed Juarez, used the variable regions obtained by them in a previous work (Juarez et al, 2013) in which analyzed the combinatorial expression of 16 inmunoglobulines against the rotavirus protein VP8 which is located in the capside region. The chosen variable region for the SIP was obtained from the combination with better activity against this epitope. This research group also demonstrated that those tomato expressing the sIgA presented the same composition than those wild type, so both were equally safe (Juarez et al, 2014).

Together with the rotavirus infection the other great cause of deaths in undeveloped countries and even more in infants ad it is shown in the UNICEF report of 2014, is the pneumonia. Being both of them the responsible of more than the 20% of children deaths.

Natural response to infection in the lower respiratory tract depends mainly in the neutrophilic granulocyte which secret several products in order to fight infection. One of these products is lactoferrin, a glicosilated protein with two homologous domains able to interact with iron ions. It is the chelating property the one which gives this protein their bacteriostatic activity (Otto et al, 1992). The bactericidal activity resides in the N-lobe of the protein, it acts agains E.coli or V.cholerae among others. Oral administrationof lactoferrin has been prove to has antimicrobial but also antiviral activity in animals models (Teraguchi et al, 2004) increasing the levels of leukocytes and cytokines as interferon gamma, interleukin 12 and 18. It also stimulates the activity of macrophages, so lactoferrin plays an important role in pathogen eradication and homeostasis maintenance in episodes of infection.

Pneumonia major cause is the infection by Streptococcus pneumoniae, causing also meningitis, septicemia and otitis media (Butler Jc et al, 1999). Mirza Shaper et al, 2004, observed that lactoferrin apoprotein (without iron ions) has bactericidal activity. They also confirmed that this activity is maintained by just the first 11 amino acids of the N-terminous domain. The oral treatment of lactoferrin in animal models has demonstrated to attenuate pneumonia by decreasing the infiltration of inflamatpry cells in lung (Kouichirou et al, 2005).

An other public health concern is hepatitis. Dr Gottfried Hirnschall, the director of the WHO programme against this viral disease, states that one again the problem in is not the weapons, we know how to prevent, control and even cure some hepatitis types. There are 1.45 million death per year caused by hepatitis, in fact the 80% of deaths produced by liver failure are caused by type B and C. However drugs do not arrive still to the one that need them most.

In 2013, the Who added the Interferon alpha to the list of essential drugs for the treatment for hepatitis. In a methanalysis performed in more than 12.000 patients pegylated interferon added to ribavirine treatment was associated with good prognosis. (Ford N et al, 2012).

Interferon alpha is a protein produced by our immune system as a defense mechanisms against viral infection or cancerigen cells. It is a glicosilated protein from the family of cytokines. It also interfers with viral replication difficulting the infection process.

In that way interferon can be applied intravenously to treat hepatitis but it can be also topic used to treat herpes or prevent the infection of any injury.

See results

Cholera Vaccine

However there is a particular disease that appear always after a natural catastrophe and in which is even more important the accessibility issue, it is the cholera. Vibrio Cholerae is one of the pathogens that mainly causes diarrhea in conjunction with rotavirus.

In high income countries colera is just a past remember of ancient pandemia. However, there are many places in the world in which it supposed a real deal. The local production of an edible vaccine will resolve this problem.

Figure 8.

The compound chosen for the vaccination is the beta subunit fron an enterotoxigenic E. coli homologous to cholera toxin (PAPER). In that way the same vaccine will prevent from both pathogenic species (enterogenic E.coli causes over 800.00 deaths per year).

See results

Bibliography

1. Beyer HM, Naumann S, Weber W, Radziwill G (2015). Optogenetic control of signaling in mammalian cells. Biotechnology Journal, 10: 273–283

2. Muller K, Siegel D., Jahnke FR., Gerrer K., Wend S., Decker EL., Reski R., Weber W., Zubriggen MD (2014). A red light-controlled synthetic gene expression switch for plant systems. Mol. BioSyst., 10: 1679-1688

3. Zhang K., Cui., B (2015). Optogenetic control of intracellular signaling pathways. Trends in Biotechnology. 33: 92-100

4. Levskaya A., Weiner OD., Lim WA. Voigt CA (2009). Spatiotemporal control of cell signaling using a light-switchable protein interaction. Nature 461: 997-1001

5. 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 9:379-384

6. Ando, R., Mizuno, H., Miyawaki, A (2014). Regulated fast nucleocytoplasmatic shuttiling observed by reversible protein highlighting. Science, 306, 1370-1373

7. Zhou XX., Chung HK., Lam AJ., Lin MZ (2012). Optical control of protein activity by fluorescent protein domains. Science 338: 810-814

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