The design of our revolutionary biological circuit enables its activation in any place and with minimal technological requirements since it is based on light-controlled systems. While chemically inducible systems for the activation of signaling pathways have been developed and applied, inherent drawbacks, such as pleiotropic effects, or toxicity along with rapid diffusion and persistence of the inducers, limit their applicability. Light-responsive systems set up the basis for the development of tools and methods to control cellular processes with high precision in time and space [1]. Several genetically encoded systems have been engineered to modulate gene expression in response to UVB, blue or red light in all kind of species, from bacteria to mammalian cells. However, less emphasis has been put to implement these ideas in plants, due to the fact that plant cells require light to gather information from their environment and to harness their energy [2].

Core components for optogenetic systems are photoactivatable proteins, such as LOV domains, phytochrome B (PhyB), cryptochrome 2 (CRY2), UV-resistance locus 8 (UVR8), Dronpa and FKF1 [3]. In order to decide which light systems are the most suited for our biological circuit we had to consider the advantages and disadvantages of each one. The method based on PhyB and its interacting factor PIF6, exhibits dimerization within seconds. Recently, Muller et al. described the first adaptation of this system to plants. The blue switch based on LOV domain and its ligand ePDZ hasn’t been yet demonstrated on plants yet, but we considered it the most suitable for our purpose.

Furthermore, we wanted to create a real light-controlled toggle switch for gene activation in plants. We took advantage of Dronpa’s capability to form heterodimers with a Dronpa mutant upon illumination with violet light, and dissociation upon illumination with cyan light.

Furthermore, we wanted to create a real light-controlled toggle switch for gene activation in plants. We took advantage of Dronpa’s capability to form heterodimerization with a Dronpa mutant upon illumination with violet light, and dissociation upon illumination with cyan light.

The red/far red toggle switch is based on the interaction between two Arabidopsis thaliana proteins, the 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 (PhyB) and allows its binding to PIF6. Far red light causes the disassociation of the proteins inactivating the genetic device.

To enable the transcriptional control of the gene of interest based on the red/far-red light transition, we fused an activation domain to PhyB and a DNA binding domain to its interactor PIF6. The chosen activation domain for the fusion with PhyB was VP16 from herpes simplex virus, while the N-terminal interaction domain of PIF6 was combined with different DNA binding domains, such as Gal4, LacI, LexA and E.

One of the main parts of our revolutionary AladDNA circuit is the blue light switch, which is based on 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), which functions as a cofactor in biological blue-light photoreceptors. 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 [3][4]. Blue light induce conformational alterations in the LOV2 domain causing a structural unwinding of its C-terminal alpha helix (referred as Jα). Upon its unwinding, a “tunable light inducible dimerization tag” (TULIP), which is an epitope tag fused to Jα, is able to interact with an engineered Erbin PDZ domain (ePDZ) [5]. For the expression of the target genes with this blue-light switch, two proteins are required. The first of them consists of the LOV2 photoactivable domain fused to a DNA-binding domain (BD). The BD binds to a specific DNA sequence located upstream a minimal promoter that drives the expression of the target gene.

The second protein is made up of the Erbin PDZ domain (ePDZ) fused to VP16, an activation domain that recruits the transcriptional machinery to the regulatory region driven the expression of the gene of interest.

As we explained above,he key step of this blue light-controlled switch is the interaction between the ePDZ domain and the peptide epitope tagged in the Jα of the LOV2 domain. A great advantage of this system is that the affinity of the interaction can be widely modulated. On the dark state, the Jα helix is not exposed to its ligand, preventing the recruitment of the ePDZ-VP16 domain, and consequently impeding the transcription of the gene of interest.

However, upon illumination with blue light, the Jα unwinds from the LOV2 domain becoming more accessible and enabling the second chimeric protein, ePDZ fused to VP16, to bind to the Jα peptide. As result of this interaction VP16 is positioned on the nearby of the regulatory region of the gene of interest recruiting the transcriptional machinery that allows its.

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 a light-inducible element for regulating gene expression. Upon the introduction of a K145N substitution, a homotetrameric complex is formed at low protein concentrations and its components monomerize upon illumination with cyan light. Generation of tandem fusions of the original monomeric form DronpaK and the mutant DronpaN enables the reversible dissociation of intramolecular DronpaN-DronpaK dimers with blue light [7]. Although this light-controlled switch has been extensively studied, as far as we know, it was never tested in plants, making its application in our project a challenge for AladDNA.

We have de novo designed and engineered two proteins for violet/cyan light-directed transcriptional activation of target genes in plants following the approach described above for AsLOV2-ePDZ.

On this case, the first protein consists of DronpaK, fused to a DNA-binding domain that binds its operator site nearby the promoter region of the target gene. The second protein is made of DronpaN fused to the VP16 domain, which acts as a transcriptional activator enabling the transcription of the target gene.

Recombinases in our circuit play an important role since they are responsible of repressing the expression of the non-chosen pathway after first light stimulus. For instance, a combination of red and blue light stimuli will activate both pathways and will end up with the synthesis of undesired products. They act by excising the CDS flanked with specific recognition sites.

Two different recombinases are needed in order to inhibit the expression of each one of them and then the promoters of these switches must have a sequence flanked with the recombinase’s recognition site close to them.

After knowing all that serine recombinases Bxb1 and PhiC31 were chosen to compose our circuit design.

Bxb1 is a protein from Mycobateriophage Bxb1’s gp35 gen. Furthermore, a CAT1 intron from Ricinus communis has been added before exon in order to increase the efficiency of the enzyme. Its function is to regulate the lysogenic cycle of the phage by integrating and excising phage’s genome in Mycobacterium smegmatis chromosome [8]. This integrase is able to recognize two different sites, one in phage’s genome (attP, “Phage attachment site”) and another in bacterial chromosome (attB, “Bacterial attachment site”)[9]. Depending on the position and sense of this sites bxb1 is able to excise or invert the target sequence. We use bxb1 as excisionase by flanking a sequence close to the promoter.

PhiC31 is another site-specific recombinase derived from a Streptomyces phage [10]. The enzyme works the same way as bxb1 since they are both serine recombinases. It recognizes two different attachments sites called also attB and attP , and also excise a sequence flanked with attB and attP sites close to the promoter.

The aim of our magic lamp is to definitely improve the accessibility of urgent needed products to those places with lack of them. Although space and mars are the places with the greatest difficulties of access, we wanted to start first by improving our surrounding, we started in the earth. There are many organization in charge of compiling information about world health problems. The World Health Organization and UNICEF have great reports about the real problems in our world. In a 2004 report they evaluate the disease incidence according to the incomes of countries, which was very informative for our decision as usually accessibility is inversely correlated with incomes.

In 2004 the WHO proposed a plan for the reduction of this differences among income countries. The next table present the expected reduction for each type of disease among the next 30 years. However this table introduced the expected results for 2015, sadly the report of this year states that the estimated reduction has not been get. UNICEF in its global action plan for the prevention and control of pneumonia and diarrhea (GAPPD) has evaluated the accessibility of the treatment to the infants with this diseases and in some cases it does not reach even the 50% of those who really need it.

Then it is clear that there’s a real health problem due to the lack of accessibility of the treatments. Those deaths are not caused by something we do not know how to fight, they are evitable deaths, and most of them affect infants under 5 years. That is why we decided to produce four different drugs capable to fight against this diseases in our magic lamp.

The first decision was something able to decrease the diarrhea disease which is the one with the highest number of affected patients in all countries.

In the UNICEF plan against diarrhea (October 2009), one of the seven action points to drastically reduce the number of deaths is the introduction of rotavirus vaccine in Africa and Asia where the burden is greatest. They have estimated that about 40% of diarrhea cases are caused by rotavirus. Cholera is the other guest in this issue but we will treat it later.

Then, introduction of a rotavirus vaccine will reduce to the half the cases of acute diarrhea. However as always it does not reach the poorest places that are the ones with greater need. In the 2012 report of UNICEF about diarrhea they insist about the importance of this vaccine and that it has not been implanted in the low income countries.

Our production of rotavirus vaccine is based in a Small Inmuno Protein (SIP). SIP construction consist in the variable region of a whole antibody with some part of its constant region. The main advantage for its production is that they are produced with just one transcript.

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 [11] in which analyzed the combinatorial expression of 16 inmunoglobulines against the rotavirus protein VP8 which is located in the capside region.

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 [13]. 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 [14] 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 [15]. Mirza Shaper et al, 2004[16], 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 inflamatory cells in lung [17].

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. [18].

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.

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 cholera 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.

The compound chosen for the vaccination is the beta subunit fron an enterotoxigenic E. coli homologous to cholera toxin [19]. In that way the same vaccine will prevent from both pathogenic species (enterogenic E.coli causes over 800.00 deaths per year).

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