Team:Valencia UPV/Results

Valencia UPV iGEM 2015

Results

Our first achievement was the design of a biological 2N decoder capable to produce 2N outputs using only N different inputs arranged in different combinations. This design will solve the accessibility issue creating a system capable to storage information and process it efficiently. Many complications were faced in the development of the idea, as the usage of a non material inductor in order to make it independent of matter transport or the need of memory to make the sequence of light pulses a determinant factor. To learn more about the circuit design click here!

Circuit biological design included two toggle switches (one from new design!) and the two recombinases structured it to make everything works. In order to prove its feasibility, the whole circuit was modeled in silico. The deterministic model showed excellent and promising results and predicted the optimal pulses sequence for each production. The modeling also solved important questions as the importance of Dronpa tetramerization in the toggle implementation. In order to know more about modeling results click here!

Once the circuit was designed and modeling gave the approval, the different control elements of the circuit needed to be tested independently. To test the performance of the light systems, we used an assay where the plant cells express the firefly luciferase luminescence protein. The luminescence measurement is highly sensitive as it can be done by a luminometer.


Toggle Switches

As we mentioned in the circuit design, essential components for our biological decoder are the blue and red light-dependent switches. These light inducible systems allow high spatiotemporal control over gene expression in order to transmit the information through the distinct circuit levels.

Red/Far-Red toggle switch

The red system is based on the protein Phytochrome B (PhyB) from Arabidopsis thaliana and its interacting factor PIF6. It is activated by light around 660 nm and it can be reversible deactivated by light around 740 nm. As previously described by Müller et al., the Red/Far-Red system was implemented in transient assays in tobacco protoplasts resulting in a strong induction. Thus, as a first step, we wanted to analyze its functionality employing distinct DNA binding domains (DBD) for controlling gene expression.

To test the function of the red light switch we adapted the macrolide gene expression tool based on the macrolide repressor protein (referred as E) in plant system with three different DBD, LexABD, LacIBD and Gal4BD and their operator sequences, OpLexA, OpLacI and OpUAS, repectively.

We used the GoldenBraid multipartite assembly reaction to create a construct composed by the P35S promoter, the DBD fused to PIF6 and the T35S terminator. As a result, we obtained three Transcriptional Units (TUs), each one containing a DBD mentioned above.

Figure x

Simultaneously, the P35S promoter, PhyB fused to the VP16 activator domain and the T35S terminator was assembled in a multipartite reaction. After a binary assembly step for each DBD, we obtained three multigenic constructs with both transcriptional units.

Figure xx

On the other hand, we needed to assemble the chimeric promoter and the gene of interest for each DBD as a transcriptional unit. The chimeric promoter is composed of 3 different parts:

  • The aforementioned DBD operators (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.

    • As we were going to perform a Luciferase assay to test the switch, the gene of interest assembled with the chimeric promoter was the Luciferase.

    Figure xx

    Both constructs, containing the red toggle switch with LexA as a binding domain, and the luciferase protein as a reporter gene were codelivered into the Nicotiana benthamiana plant (see agroinfiltration) and after two hours protoplasts were made (see protoplasts obtaining!).

    The firefly luciferase reporter was followed over a time-course of 24 h by quantifying its luminescence. (Figure 1)

    Figure 1. kinetics of the Red/Far-Red 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 660 nm red 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 in time.

    As it can be observe, samples transfected only with the chimeric promoter and the luciferase gene construct showed basal expression, which it is maintained during all time course. In contrast, samples that continuously received 660 nm illumination began to significantly increase the luciferase luminescence after 9 hours. However, they showed a decrease in the luminescence signal after 16 hours.

    Although the previous figure 1 showed that there is an increase in luciferase expression in N. benthamiana protoplasts, we wanted to go further and explore if this system would also work in transiently transfected N. benthamiana plants.

    To do so, the construct containing the E-PIF6 and PhyB-VP16 transcriptional units was co-transformed, together with the chimeric promoter nearby the luciferase gene transcriptional unit, by Agrobacterium into N. benthamiana leaves. After 2 days, samples were taken and the luciferase expression was measured (Figure 2).

    Figure 2. kinetics of the Red/Far-Red light-regulated gene expression system in N. benthamiana leaves for light-responsive expression of firefly luciferase. Two days after transformation, discs were made and samples were illuminated with 660 nm red light and in white light during 24 hours. Control discs were incubated in the dark for the entire experiment. Firefly luciferase luminescence was quantified at the indicated points in time

    Blue switch

    The blue light-dependant gene expression system based on AsLOVpep and ePDZ interaction would also be responsible to transmit the information through the decoder (see our circuit model!). As for the previous described red switch, we also wanted to analyze its functionality in plants, employing the same DNA binding domains (DBD) to control gene expression, i.e., LexABD, LacIBD and Gal4BD and their operator sequences, OpLexA, OpLacI and OpUAS, repectively.

    Employing the GoldenBraid assembly system we were able to create a construct composed by the Cauliflower Mosaic Virus promoter (P35S), the DBD fused to AsLOVpep and the T35S terminator. We obtained three TUs, each one containing a distinct DBD. Simultaneously, it was also assembled in a multipartite reaction a TU composed by the P35S promoter, ePDZ fused to the VP16 transcriptional activator and the T35S terminator.

    Figure x

    Finally, after a binary assembly step with the two previously described TUs, we obtained three multigenic constructs.

    Figure x

    As we were going to perform a Luciferase assay to test the switch, the luciferase reporter gene, together with the control Renilla and P19 genes, was available as a single multigenic construct in the GoldenBraid collection.

    Figure x

    Finally, after one more GoldenBraid binary assembly step, we obtained three multigenic constructs.

    In order to test the blue light-dependant switch, we perform a luciferase assay. For this purpose, the construct containing the LacI DNA binding domain was used to transform N. benthamiana leaves. After two days, discs were made from the leaves. And the expression levels of the luciferase reporter gene were measured by a luminometer (Figure 3). As a negative control, plants were infiltrated with LacI operator fused to the luciferase and renilla genes.

    Figure 3. Luciferase assay showing the luciferase/renilla ratios of the AsLOV-ePDZ blue light-regulated gene expression system in N. benthamiana leaves. Two days after Agrobacterium-mediated transformation, discs were made and samples were illuminated with 470 nm blue light and in white light during 48 hours. Control discs were incubated in blue light for the entire experiment. Firefly luciferase luminescence was quantified at the indicated points in time.

    It can be observed that the blue switch composed mainly by AsLOVpep and ePDZ appears to be active upon illumination with blue light and white light, when comparing it with the negative control at 48 hours. A greater induction occurs in the samples illuminated with light than in those samples illuminated with blue light. Further investigation and experiments should be done to confirm its activation in plants.

    Violet/Cian Toggle Switch

    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

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.

Bxb1

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).

Figure 1. Expression levels of GFP in N. benthamiana leaves. A) Agrobacterium-mediated transformation with the multigenic construct BBa_K1742009. B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.

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.

PhiC31

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).

Figure 1. Expression levels of GFP in N. benthamiana leaves. A) Agrobacterium-mediated transformation with the multigenic construct BBa_K1742013. B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.

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.

Drug production

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.

Rotavirus Sip

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.

Figure 1.SIP simplificated structure

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.

Figure 2.Detection of anti-rotavirus SIP

Lactoferrin

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.

Figure 3. Lactoferrin 3D Structure

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.

Interferon

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.

Figure 4.Interferon Detection by inmuno asssay

Cholera Vaccine

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>

Figure 5.Inmuno detection of colera vaccine produced in plant leaves

Conclusion

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.

Section 5

The circuit was designed for plant implementation so we wanted to asses which species would be better for that porpoise. In order to achive that we transciently transformed three different plant species:

Soya Beans

Soya would be an ideal chasis as they grow very fast and are known worldwide. They are approve by human consumption and what is more important germination only last one day! In our tray to agroinfiltrate them we firstly do it with comassie colorant in order to see if liquid when seedling are submitted to vacuum. As it is observed in Figure 1, the external part of the epidermis is coloured but when sections were analyzed the inner part of the stem was not coloured.

FOTO

Although the colorant did not work we tried to agroinfiltrate with agrobacterium with fluorescent protein construction (DsRed) by vacuum. Seedling were observed under the Leica magnifier but no fluorescence was observed.

Sunflowers

Sunfloers are very beautifull plants known by their appereance in many famouse paintings. Sunflowers produce edible fruits with great consumption in Spanish population, however there is no tradition of sunflowers in the places where AladDNA is needed. In spite of that, the germination process is slower than soya but still short (4-5 days).

Seedlings were agroinfiltrated with DsRed fluorescent protein. Some signal was observed after three days of infiltration but very high adquisitions times were needed in order to make it visible.

Figure 1.Fluorescence observed in sunflowers after agroinfiltration with DsRed

Spinach

Spinach are the terror of childs at lunch time, but they are also the favourite Popeye food. So if spinachs can turn Popeye into a strong man why not use spinach to save their lives?

They have big leaves so high expression levels could be reached. However, they present a big problem, they last 10 days in order to germinate and they were more delicated than the others.

We agoinfiltrated this seeedlings with DrRed we could see a huge amount of it, mainly concentrated in the rowels. Three days after that, samples were evaluated in the magnifier and fluorescence was observed (Figure 3).

Figure 1.Fluorescence observed in spinach after agroinfiltration with DsRed

Biorreactor

In this part we also wanted to test the method more adeccuated for a fast and easy germination. With this porpoise, Control seed were grown in petri dishes with cotton and seed were kept in different devices with different characteristics. Some seed were submerged in water, others were kept with very very little water in the bottom of the cup and others were subjected into a device capable to keep them in the water surface. This last device was the one with better results not only for germination but also for agitation. Agitation was also tested because in the magic lamp we need it to adquire a complete irradiation of light by switches. Then the agitation system of our lamp also works in the same way as our Chocolate cup maker .

Figure 1.Fluorescence observed in spinach after agroinfiltration with DsRed

Section 5

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