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Our “de novo” designed blue toggle switch is based in DronpaK, a blue light–sensitive protein able to change its conformation depending on the wavelength irradiated. Upon the introduction of a K145N substitution, a homotetrameric complex is formed. When it is irradiated with 390 nm the complex monomerizes (switch on) and when irradiated with 490nm they tetramerizes again (switch off).

In order to test the functionality of our ”de novo” designed blue light-dependent toggle switch in plants; we used GoldenBraid multipartite assembly to create a construct composed by the P35S promoter, the DBD fused to dronpaK and the T35S. Since we used three different binding domains, we obtained three different Transcriptional Units. Simultaneously, the P35S promoter, DronpaK fused to the activator domain VP16 and the T35S terminator were assembled in a multipartite reaction. After a binary assembly stapp for each DBD, we obtained three multigenic constructs with both transcriptional units.

Moreover, we needed to assemble the chimeric promoter and the gene of firefly Luciferase, since we studied the luminescence of each sample with the luminometer.

Next, we needed to ligate the chimeric promoter and the gene of interest for each DBD as a transcriptional unit. This chimeric promoter is composed of 3 different parts:

• The DBD operators of the binding domains above mentioned (OpLexA, OpLacI and OpUAS), which are the binding sites of the corresponding binding domain.
• A minimal promoter, miniP35S(-60), required for starting transcription.
• The 5’-UTR region of the tobacco mosaic virus (TMV), called omega sequence. It functions as a translational enhancer in plants.

The gene of interest assembled with the chimeric promoter was the Luciferase since we were going to perform a Luciferase assay to test the switch, Both constructs, containing the blue toggle switch with LexA as a binding domain, and the luciferase protein as a reporter gene were made (see protoplasts obtaining).

The firefly luciferase reporter was followed over a time-course of 24h by quantifying its luminescence FigureX.

Figure X. kinetics of the near UV/Blue light-regulated gene expression system in N.benthamiana protoplasts.Protoplasts were obtained from Agrobacterium-transfected leaves for light-responsive expression of firefly luciferase. Two hours after transformation., protoplast samples were illuminated with 390nm UV light and in white light during 24 hours. Control cells were incubated in the dark for the entire experiment. Firefly luciferase luminescence was quantified at the indicated points.

Moreover, a sample of protoplasts transformed with the entire construction of the blue toggle was observed in a confocal microscope irradiating with wavelengths of 405 nm (Dronpa activation), 488nm (Dronpa deactivation).



Figure 2. Fluorescence of Dronpa protein in N. benthamiana protoplasts.Protoplasts were obtained from Agrobacterium-transfected leaves. Two hours after transformation, protoplast sample was collected and observed in a confocal microscope As it can be observed when applying a 405nm laser, fluorescence inside nucleus is observed, besides some fluorescence appears as time goes by. Nevertheless, after irradiating with 488nm the protoplast has lost the majority of its cytoplasm fluorescence as well as its nucleus fluorescence. Finally, after applying a 405nm laser again nucleus fluorescence is reestablished.

Recombinases are one of the key points for our circuit performance. Experiments to prove their correct working in the same way expected in the circuit was evaluated with trasncient expression in Nicotiana.

Since we wanted to prove that the codon-optimized recombinase bxb1 works in Nicotiana Benthamina, we designed a reporter element that consists on Cauliflower Mosaic Virus terminator (T35S) flanked with bxb1 attachment sites (attB:T35S:attP:omegaUTR) which allows recognition by the recombinases and excision of the fragment flanked.

After constructing a composite formed by bxb1 recombinase with its reporter element, a GFP coding sequence and T35S (P35S:bxb1:T35S-P35S-attB:T35S:attP:omegaUTR-GFP:T35S), this construct was transformed into Agrobacterium tumefaciens and agroinfiltrated in N.Benthamiana leaves by agroinfiltration. In addition, this construction was coinfiltrated with P19, a viral silencing repressor in order to increase the signal of the samples. Moreover we agroinfiltrated another plant just with the reporter ligated with GFP as negative control (P35S-attP:T35S:attB:omegaUTR-GFP:T35S).

Five days after the infiltration some discs leaves were taken and observed with a confocal microscope. As it could be seen in the images above, bxb1 works in Nicotiana Benthamiana although negative control presents some basal expression.

Since we wanted to prove that the codon-optimized recombinase phiC31 works in Nicotiana Benthamina, we designed a reporter element that consists on Cauliflower Mosaic Virus terminator (T35S) flanked with phiC31 attachment sites (attP:T35S:attB:omegaUTR) which allows recognition by the recombinases and excision of the fragment flanked .

After constructing a composite formed by phiC31 recombinase with its reporter element, a GFP coding sequence and T35S (P35S:phiC31:T35S-P35S-attP:T35S:attB:omegaUTR-GFP:T35S), this construct was transformed into Agrobacterium tumefaciens and agroinfiltrated in N.Benthamiana leaves by agroinfiltration. In addition, this construction was coinfiltrated with P19, a viral silencing repressor in order to increase the signal of the samples. Moreover we agroinfiltrated another plant just with the reporter ligated with GFP as negative control (P35S-attP:T35S:attB:omegaUTR-GFP:T35S).

Five days after infiltration we take some discs of leaves and observe them with a confocal microscope. As it could be seen in the images above, results were positive, phiC31 works in Nicotiana Benthamiana although the negative control presents some basal expression since some fluorescent cells were spotted.

The concrete aim of AladDNA in this occasion is the production of one of the most needed drugs in the remote areas of the world. The production of our compounds was achive by transcient expression in Nicothiana Benthamiana leaves.

The sequence construction was kindly given to us by Juarez P. It was created from the variable region of an IgA created by them against rotavirus. However as the structure of small inmunoproteins contains some constant regions, two different constructions were given to us with different fragments of the constant region. The objective of this two structures is to achive which one is more affine to the capside protein of the virus as the affinity can vary as the conformational structure can change.

Two leaves of Nicotiana were agroinfiltrated for each SIP and after three days samples were collected and a western blot inmuno assay was performed. Western membranes were probed with a labelled antibody against anti-rotavirus IgA.

As it can be observed in the inmunodetection assay, only the SIP with the CH2 constant fragment was recognized by the anti-IgA against rotavirus. In the results there are two detection bands for the CH2 SIP the one with higher molecular weight corresponds to the complete protein produced by the plant. However there is a second band with lower molecular weight caused by the protein degradation that occurs also in the plant.

The lactoferrin construction was designed by us. This protein consist in two homologous domains with iron chelant capability. Although it is known that the desired antimicrobial effect is caused by the N-terminous domain, the lactoferrin has many other beneficial effects as modulate the immune system. Therefor we decided to produce the whole protein in our device.

For that purpose, we ordered synthesis the whole CDS in two different gBlocks as its length impossibility the synthesis in one. We designed the gBlocks in order to be able to assemble them by Golden Braid assembling using a plant promoter, a signal peptide for secretion and a plant terminator. The construction of the whole CDS was achived and probed by digestion with restriction enzymes. The final construction with the transcriptional unit did not get the expected deadline so it was not tested in plants.

The interferon construction was assemble from basic parts from the Golden Braid 2.0 collection. After the assembling of the whole transcriptional unit with the promoter, a signal peptide for secretion, the CDS and a terminator; it was transformed to Agrobacterium in order to produce a transcient expression in Nicotiana.

Two leaves were infiltrated and collected three days after that in order to perform a western blot inmuno assay. The probe was performed with an anti-His antibody (CDS was His tagged) and a labeled secondary antibody. As it can be seen in Figure 3, there is a band that correspond to the expected molecular weight of Interferon.

In order to produce a Cholera Vaccine we looked for edible vaccines capable to be produced in plants. In this research we found a study from ___ in which a cholera vaccine was produced immunizing with a labile entherobacteria toxin from E. coli. This LTB is produced in plants by ___. Using their sequence we order to synthesis a gBlock and with golden braid assembling we obtained a transcriptional unit for plants.

The LTB was also tagged with His so it was detected by inmuno assay with anti His and a labeled secondary antibody. We obtained a huge expression of this protein inside the plant at the expected molecular weight as it is observed in the Figure 4. /p>

Drug production using plants as biofactories, is a real achievement. This chasis allows high production rates in an eco-friendly manner. It is also very interesting specifically in case of biodrugs as they might need some post translational modifications imposible to be achived by other species. An other huge advantage in plant productions is that they are free of human and animal viruses and diseases so there are less risks in the purification!

We achived the constructions of three from four of the designed transcriptional units for drug production. The three constructions were finely expressed in Nicothiana. However further studies are required in order to prove the biological activity of all of them.

Hello world

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