iGEM 2017 — Team Valencia_UPV

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# Team:Valencia UPV/Notebook

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* TEAM
  + [Team](https://2017.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2017.igem.org/Team%3AValencia_UPV/Attributions)
* PROJECT
  + [Description](https://2017.igem.org/Team%3AValencia_UPV/Description)
  + [Applied Design](https://2017.igem.org/Team%3AValencia_UPV/Applied_Design)
  + [Collaborations](https://2017.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Achievements](https://2017.igem.org/Team%3AValencia_UPV/Achievement)
  + [Safety](https://2017.igem.org/Team%3AValencia_UPV/Safety)
* WET LAB
  + [Biological Design](https://2017.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2017.igem.org/Team%3AValencia_UPV/Experiments)
  + [Protocols](https://2017.igem.org/Team%3AValencia_UPV/Protocols)
  + [Results](https://2017.igem.org/Team%3AValencia_UPV/Results)
  + [Notebook](https://2017.igem.org/Team%3AValencia_UPV/Notebook)
  + [Contribution](https://2017.igem.org/Team%3AValencia_UPV/Contribution)
  + [PlantSynBio](https://2017.igem.org/Team%3AValencia_UPV/Plant)
  + [Parts](https://2017.igem.org/Team%3AValencia_UPV/Parts)
* DRY LAB
  + [Modeling](https://2017.igem.org/Team%3AValencia_UPV/Model)
  + [Software](https://2017.igem.org/Team%3AValencia_UPV/Software)
  + [Hardware](https://2017.igem.org/Team%3AValencia_UPV/Hardware)
  + [Graphic Design](https://2017.igem.org/Team%3AValencia_UPV/GraphicDesign)
* HUMAN PRACTICES
  + [Outreach](https://2017.igem.org/Team%3AValencia_UPV/HP/Silver)
  + [Integrated and gold](https://2017.igem.org/Team%3AValencia_UPV/HP/Gold_Integrated)

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### NOTEBOOK

[PDF](https://static.igem.org/mediawiki/2017/5/5f/NotebookPdfDefinitivoValencia.pdf)

## INTRODUCTION

After months of frenetic activity, our project was finally finished and we felt very excited. Although the path was difficult, we pushed ahead and gave our best. As a result, we are very proud of presenting ChatterPlant and we are excited to share our complete work during all this amazing summer.

**Valencia UPV iGEM** Team

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# Team:Valencia UPV/Experiments

[![](https://static.igem.org/mediawiki/2017/d/da/LogotipoChatterPlantNavBar.png)](https://2017.igem.org/Team%3AValencia_UPV)

* TEAM
  + [Team](https://2017.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2017.igem.org/Team%3AValencia_UPV/Attributions)
* PROJECT
  + [Description](https://2017.igem.org/Team%3AValencia_UPV/Description)
  + [Results](https://2017.igem.org/Team%3AValencia_UPV/Results)
  + [Applied Design](https://2017.igem.org/Team%3AValencia_UPV/Applied_Design)
  + [Collaborations](https://2017.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Achievements](https://2017.igem.org/Team%3AValencia_UPV/Achievement)
  + [Safety](https://2017.igem.org/Team%3AValencia_UPV/Safety)
* WET LAB
  + [Biological Design](https://2017.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2017.igem.org/Team%3AValencia_UPV/Experiments)
  + [Protocols](https://2017.igem.org/Team%3AValencia_UPV/Protocols)
  + [Notebook](https://2017.igem.org/Team%3AValencia_UPV/Notebook)
  + [Contribution](https://2017.igem.org/Team%3AValencia_UPV/Contribution)
  + [PlantSynBio](https://2017.igem.org/Team%3AValencia_UPV/Plant)
  + [Parts](https://2017.igem.org/Team%3AValencia_UPV/Parts)
* DRY LAB
  + [Modeling](https://2017.igem.org/Team%3AValencia_UPV/Model)
  + [Software](https://2017.igem.org/Team%3AValencia_UPV/Software)
  + [Hardware](https://2017.igem.org/Team%3AValencia_UPV/Hardware)
  + [Graphic Design](https://2017.igem.org/Team%3AValencia_UPV/GraphicDesign)
* HUMAN PRACTICES
  + [Outreach](https://2017.igem.org/Team%3AValencia_UPV/HP/Silver)
  + [Integrated and gold](https://2017.igem.org/Team%3AValencia_UPV/HP/Gold_Integrated)

Home • Wet Lab •

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### EXPERIMENTS

[PDF](https://static.igem.org/mediawiki/2017/9/9c/ExperimentsDefinitivoValencia.pdf)

#### INDEX

* [Introduction](#intro)
* [Phic31 Performance](#Phic31)
* [Gp3 Performance-Reporter (L X R) - 1](#procedures1)
* [Gp3 Performance-Reporter (L X R) - 2](#procedures2)
* [Phic31 Performance (P X B)](#Phic31Per)
* [Phic31 Performance-Promoters](#Phic31promot)
* [Characterization Of Phic31 Performance](#Phic31carac)
* [PVX](#PVX)
* [Optogenetic–Controlling the Genetic Expression with Light](#optogenetic)
* [Optogenetic 2.0](#optogenetic2)
* [GP3 Performance](#GP3)
* [Metabolic Rate](#metabolic)
* [Chromoproteins](#chromoproteins)
* [Chromoproteins-PVX](#chromoPVX)
* [Stress-Promoter PR-1A](#stress)

## INTRODUCTION

Experimental design is the heart of the iGEM competition, intelligent development of how the initial problem proposed is going to be solved involves different approaches from the different disciplines that our team is composed by. In this section, we show how we obtained the solutions used for the validation of the initial ideas

## PHIC31 PERFORMANCE

#### **Objective**

Testing the performance of phiC31 recombinase using the register assembly construct with the attachment sites PxB.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/4/4c/Experimento1Valencia.png)

## GP3 PERFORMANCE - REPORTER (L X R) - 1

#### **Objective**

Testing the performance of gp3 using the register assembly construct with the recombined attachment sites LxR.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/b/b2/Experimento2_3Valencia.png)

*Figure 2. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of a fluorescent protein. It represents the negative control of our experiment. b) Genetic construct that allows constitutive expression of phiC31 in the plant. c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of RDF under a strong promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (F.2a and c) were 0,02, the optical density of PhiC31 construct (F.2b) was 0,1 and the optical density of gp3 construct (F.2d) was 0,05 and 0,15. A triplicate sampling of different plants was performed from 36h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly construct (Fig. 2a) was agroinfiltrated together with recombinase (Fig. 2c) on 21th August at 12:00h. Positive and negative controls (Fig. 2b and Fig. 2a) were also infiltrated. After 48h post-infiltration (23th August), positive and negative control were sampled and RDF (Fig. 2d) was agroinfiltrated. After 36h, leaves were sampled every eight hours finishing on 25th August at 08:00h. At this point, positive and negative controls were also sampled. Overall, ten points of samples were taken in the assay after recombinase were agroinfiltrated.

## GP3 PERFORMANCE - REPORTER (L X R) - 2

#### **Objective**

Testing the performance of gp3 using the register assembly construct with the recombined attachment sites LxR.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/b/b2/Experimento2_3Valencia.png)

*Figure 3. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of a fluorescent protein. It represents the negative control of our experiment. b) Genetic construct that allows constitutive expression of phiC31 in the plant. c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of RDF under 35s promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (F. 3a and c) were 0,02, the optical density of PhiC31 construct (F.3b) was 0,05 and the optical density of gp3 construct (F.3d) was 0,20 and 0,35. A triplicate sampling of different plants was performed from 36h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly construct (Fig. 3a) was agroinfiltrated together with recombinase (Fig. 3c) on 3th August at 12:00h. Positive and negative controls (Fig. 3b and Fig. 3a) were also infiltrated. After 48h post-infiltration (7th September), positive and negative control were sampled and RDF (Fig. 3d) was agroinfiltrated. After 36h, leaves were sampled every eight hours finishing on 11th September at 20:00h. At this point, positive and negative controls were also sampled. Overall, six points of samples were taken in the assay after recombinase were agroinfiltrated.

## PHIC31 PERFORMANCE (P X B)

#### **Objective**

Testing the performance of phiC31 recombinase using the register assembly construct with the attachment sites PxB in order to characterize luciferase dynamics.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/1/16/Experimento10Valencia.png)

*Figure 4. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 4a and Figure 4c) was 0,02 and the optical density of PhiC31 construct (Figure 4b) was 0,1. A triplicate sampling of different plants was performed from 36h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly constructs (Fig. 4a and 4b) were agroinfiltrated on 3th August at 12:00h. After 48h post-infiltration (7th September), positive and negative control were sampled and the recombinase was agroinfiltrated. After 36h, leaves were sampled every 24 hours finishing on 11th September at 20:00h. At this point, positive and negative controls were also sampled. Overall, four points of samples were taken in the assay after recombinase were agroinfiltrated.

## PHIC31 PERFORMANCE - PROMOTERS

#### **Objective**

Testing the performance of phiC31 recombinase using the register assembly construct with the attachment sites PxB in order to characterize luciferase dynamics.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/a/a4/Experimento5Valencia.png)

*Figure 5. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a weak promoter. e) Transcriptional unit for the expression of the Firefly Luciferase under the control of a strong promoter. f) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 5a and Figure 5c) was 0,02 and the optical density of PhiC31 construct (Figure 5b) was 0,01. A triplicate sampling of different plants was performed in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly constructs (Fig. 5a and 5b) and the controls (Fig. 5e and 5f) were agroinfiltrated on 19th September at 12:00h. After 24h post-infiltration (21th September), leaves were sampled at 18:00h. After 48h, all leaves were also sampled. Overall, two points of samples were taken in the assay.

## CHARACTERIZATION OF PHIC31 PERFORMANCE

#### **Objective**

Testing the performance of phiC31 recombinase using the register assembly construct with the attachment sites PxB in order to characterize luciferase dynamics under a weak promoter.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/3/38/Experimento6Valencia.png)

*Figure 6. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when ΦC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a weak promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Transcriptional unit for the expression of the Firefly Luciferase under the control of a strong promoter. e) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 6a and Figure 6c) was 0,02 and the optical density of PhiC31 construct (Figure 6b) was 0,01 AND 0,005. A unique sampling of the same plant was performed.

#### **Timeline**

Register assembly constructs (Fig. 6a and 6c) and the controls (Fig. 6e and 6f) were agroinfiltrated on 29th September at 18:00h. After 12h post-infiltration (30th September), leaves were sampled every 6 hours finishing on 1st October at 08:00h.

## PVX

#### **Objective**

Testing the viral systemic movement performed by Potato Virus X (PVX).

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/d/d4/Experimento7Valencia.png)

*Figure 7. a) Graphic representation of PVX-dsRED- Express Vector. When the viral vector is being produced, the fluorescent protein (dsRED) will be expressed.*

#### **Method**

An agroinfiltration in plant roots was performed in order to test the systemic movement of this viral vector. To do so, the construct containing the fluorescent protein (dsRED) was transformed by Agrobacterium into N. benthamiana leaves and roots. (Figure 7a). The final Optical Density of the genetic construct was 0,1.

#### **Timeline**

One week after transformation, plants were observed at the magnifier in order to check the fluorescence in the aerial part of the plant.

## OPTOGENETIC – CONTROLLING THE GENETIC EXPRESSION WITH LIGHT

#### **Objective**

The genetic expression control with red-light. The main objective of the following experiment is testing the biological performance of the red-far red switch when it is irradiated with red light.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/b/b2/Optogenecircuit.png)

*Figure 8. a) Graphic representation of the first part of the designed genetic circuit Red/ far-red toggle-switch, when activated, will activate luciferase transcription. To do so, PIF6 is fused to a DNA-binding domain (E), which binds to Etr8 operator. Moreover, PhyB is fused to an activator domain (VP64) and a nuclear location sequence (NLS). b) Conformation of the proteins which belong to genetic toggle switch. c) When irradiated with 660nm light, PhyB changes its conformation and this complex is recruited to PIF6 at the promoter site. The polymerase III will recognize the activation domain and the transcription will begin. Only upon absorption of a far-red photon the interaction between PhyB and PIF6 is terminated, resulting in a shut-off of gene expression.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. To do so, the construct containing the E-PIF6 and PhyB-VP16 transcriptional units was transformed, together with the chimeric promoter nearby the luciferase gene transcriptional unit, by Agrobacterium into N. benthamiana leaves (Figure 8a and 8c). The final Optical Density of optogenetic constructs was 0,1. Several treatments were performed in order to test the toggle switch. Both control and sample discs were incubated in the dark during the first 36h. After that, two samples were illuminated with 660 nm red light (hardware link), two other samples plants and two controls were illuminated with white light and finally, the last two samples and controls were incubated in the dark for the entire experiment.

#### **Timeline**

Optogenetic experiment was performed for 24 hours and samples were taken at 0 hrs, 3 hrs, 6 hrs, 9 hrs, 14 hrs and 23 hrs. Sampling of control plants were made at 0 hrs and 23 hrs.

## OPTOGENETIC 2.0

#### **Objective**

The genetic expression control with red-light. The main objective of the following experiment is testing the biological performance of the red-far red switch when it is irradiated with red light.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/b/b2/Optogenecircuit.png)

*Figure 9. a) Graphic representation of the first part of the designed genetic circuit Red/ far-red toggle-switch, when activated, will activate luciferase transcription. To do so, PIF6 is fused to a DNA-binding domain (E), which binds to Etr8 operator. Moreover, PhyB is fused to an activator domain (VP64) and a nuclear location sequence (NLS). b) Conformation of the proteins which belong to genetic toggle switch. c) When irradiated with 660nm light, PhyB changes its conformation and this complex is recruited to PIF6 at the promoter site. The polymerase III will recognize the activation domain and the transcription will begin. Only upon absorption of a far-red photon the interaction between PhyB and PIF6 is terminated, resulting in a shut-off of gene expression.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. To do so, the construct containing the E-PIF6 and PhyB-VP16 transcriptional units was transformed, together with the chimeric promoter nearby the luciferase gene transcriptional unit, by Agrobacterium into N. benthamiana leaves (Figure 9a and 9c). The final Optical Density of optogenetic constructs was 0,1. Several treatments were performed in order to test the toggle switch. Both control and sample discs were incubated in the dark during the first 36h. After that, two samples were illuminated with 660 nm red light (hardware link), two other samples plants and two controls were illuminated with white light and finally, the last two samples and controls were incubated in the dark for the entire experiment.

#### **Timeline**

Optogenetic experiment was performed for 50 hours and samples were taken at 0 hrs, 3 hrs, 8 hrs, 11 hrs, 13 hrs, 16 hrs, 19 hrs, 23 hrs, 25 hrs, 25hrs, 27 hrs, 29 hrs, 31 hrs, 34 hrs and 50 hrs. Sampling of control plants were made at 0 hrs, 24 hrs, 34 hrs and 50 hrs hrs.

## GP3 PERFORMANCE

#### **Objective**

Testing the performance of gp3 using the register assembly construct with the recombined attachment sites LxR.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/a/a0/Experimento9Valencia.png)

*Figure 10. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of a fluorescent protein. It represents the negative control of our experiment. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a weak promoter (Pnos Promoter) c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites (attB and attP). It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of RDF under 35s promoter (strong promoter). e) Transcriptional unit for the expression of the Firefly Luciferase under the control of a strong promoter. f) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (F. 10a and c) were 0,01, the optical density of PhiC31 construct (F.10b) was 0,005 and the optical density of gp3 construct (F.10d) was 0,10 and 0,20. A unique sampling of the same plant was performed from 36h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly construct (Fig. 10a) was agroinfiltrated together with recombinase (Fig. 10c) on 12th October at 18:00h. Positive and negative controls (Fig. 10a and Fig. 10c) were infiltrated together with the internal controls (Fig. 10e and 10f). After 48h post-infiltration (14th October), the RDF (Fig. 10d) was agroinfiltrated letting a control plant without being agroinfiltrated. After 36h, leaves were sampled every twelve hours finishing on 18th October at 20:00h. At this point, positive and negative controls were also sampled. Overall, six points of samples were taken in the assay after recombinase were agroinfiltrated.

## METABOLIC LOAD

#### **Objective**

Testing the metabolic rate of cells that can affect recombinase performance from the molecular to the organismal level.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/b/b6/MetabolicLoadValencia.png)

Figure 11. a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a weak promoter (Pnos promoter).

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 11a) was 0,02 and the optical density of PhiC31 construct (Figure 11b) was 0,01 and 0,2. A triplicate sampling of different plants was performed from 48h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Register assembly construct (Fig. 11a) was agroinfiltrated together with the recombinase (Fig.11b) letting a control plant without it on 20th October at 12:00h. After 48h post-infiltration (22th October) all plants were sampled. After 96h post-infiltration, leaves were also sampled. Overall, two points of samples were taken in the assay after recombinase were agroinfiltrated.

## CHROMOPROTEINS

#### **Objective**

Testing the performance chromoproteins in plants.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/5/5c/Experimento11Valencia.png)

*Figure 12. a) Graphic representation of the construct comprised by the chromoprotein AmilCP under a strong promoter (35s Promoter) b) Graphic representation of the construct comprised by the chromoprotein eforRed under a strong promoter (35s Promoter).*

#### **Method**

An agroinfiltration was performed in order to test chromoprotein gene expression. The final Optical Density of reporter constructs (F. 12a and b) were 0,1.

#### **Timeline**

One week after transformation, plants were observed at the magnifier in order to check the color protein in plant leaves.

## CHROMOPROTEINS-PVX

#### **Objective**

Testing the performance chromoproteins in plants.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/e/e5/Experimento12Valencia.png)

*Figure 13. a) Graphic representation of the viral vector construct comprised by the chromoprotein AmilCP. When the viral vector is being produced, the color protein (AmilCP) will be expressed. b) Graphic representation of the construct comprised by the chromoprotein eforRed. When the viral vector is being produced, the color protein (eforRed) will be expressed.*

#### **Method**

An agroinfiltration was performed in order to test chromoprotein gene expression. The final Optical Density of reporter constructs (F. 13a and b) were 0,1.

#### **Timeline**

One week after transformation, plants were observed at the magnifier in order to check the color protein in plant leaves.

## STRESS-PROMOTER PR-1A

#### **Objective**

Testing the performance of the stress-promoter under several treatments.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/1/1e/Experimento13Valencia.png)

*Figure 14. a) Transcriptional unit for the expression of the Firefly luciferase under the control of a stress promoter. b) Transcriptional unit for the expression of the Firefly luciferase under the control of a stress promoter when Salicylic acid is being synthesized. c) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter.*

#### **Method**

An agroinfiltration and subsequent luciferase assay were performed in order to study gene expression at transcriptional level. The final Optical Density of genetic constructs was 0,1. A triplicate sampling of different plants was performed from 48h post infiltration onwards in order to take account for biological variability due to unknown or uncontrollable conditions.

#### **Timeline**

Genetic constructs were agroinfiltrated and after 70h post-infiltration, all plants were sampled.

## TEV

#### **Objective**

Testing the performance of Ros1 using TEV as a viral vector.

#### **Plant Chassis**

Nicotiana benthamiana

#### **Parts**

![](https://static.igem.org/mediawiki/2017/7/71/Figura_TEV_Valencia.png)

*Figure 15. a) Graphic representation of the viral vector construct comprised by the ROS1 gene. When the viral vector is being produced, the protein ROS1 will be expressed*

#### **Method**

An agroinfiltration was performed with construction 15 a) in order to test ROS1 expression over the plant.

#### **Timeline**

One week after transformation, plants were observed in order to check the color change in plant leaves.

×

#### LUCIFERASE ASSAY

This procedure is done with the Promega; kit (Dual-Luciferase Reporter Assay System). Using Agrobacterium tumefaciens as a vehicle to insert the desired devise, it is necessary insert it into a leaf of N.benthamiana by a direct injection. This method is known as Agroinfiltration. The next step is letting infiltrated leafs for two or three days depending on how the experiment is programmed.

After two days post infiltration, users can get leaf disks from N.benthamiana using a hole punch. It is recommended to take the maximum agroinfiltrated area avoiding plant nerves. Leaf discs are put in a specific plate depending on the light condition requirements. Different samples are taken during the next two days after discs were made and immediately they are put in liquid nitrogen and stored at -80ºC. The steps to follow are:

1. The Passive lysis buffer 1x is prepared. Each disk of leaf needs 200µl. The passive lysis buffer is stored at 5X so it must be diluted with distilled water. Place it on the ice besides the LUCII substrate and the STOP solution.
2. Cut off two little leaf disks of approximately 0.8 cm and put it into an Eppendorf tube. Immediately, freeze it with liquid nitrogen to avoid the deterioration of vegetal material. Grind the freeze sample using the metabolomics robot. Put the samples on ice.
3. Add 150µl of passive lysis buffer 1x to each Eppendorf tube.
4. Vortex gently and centrifuge 13200rpm during 15 minutes at 4ºC. While switch on the luminometer.
5. Dilute 2:3 the extracts on a new Eppendorf tube. Add 36µl of Passive lysis buffer 1x and 24µl of sample.
6. Take an optimal plate to use in the luminometer. Fill luminometer wells with 40 µl of LUCII which is stored at -20ºC.
7. 10 µl of sample is added in each well. Wait 10min. During this time turn on and configure the luminometer.
8. Measure luciferase activity.
9. Prepare 40 µl/well of Dual Glo 1x (STOP solution + substrate). The substrate is at 50x concentration and stored at –20ºC.
10. When the first luciferase measure is done, it is necessary to add 40 µl of Dual Glo into each well. Let it rest during 10 min.
11. Measure the Renilla activity.
12. Take the obtained information and analyze it.

Things to keep in mind for the next experiment: The luminometer (machine to measure the luminescence) has to be ready before adding the reagents to the samples because it needs 10min to be ready. Set the timer (10min) with the first sample of luciferase and add the reactant to the other samples as quick as possible.

×

#### AGROBACTERIUM INFILTRATION

To carry out transient expression experiments, the method described by Orzaez et al. was followed (Orzaez et al. 2009). First of all, Agrobacterium tumefaciens GV3101 cultures incubated at 28ºC overnight were centrifuged at 3000rpm for 15 minutes. Cultures were pelleted and resuspended in 5mL of agroinfiltration solution (MES 10mM, pH 5.6, MgCl2 10mM and acetosyringone 200mM diluted with dimethyilsulfoxide (DMSO)).

Once pellet was resuspended, cultures were protected from light and incubated at room temperature for 2h under stirring conditions. Afterwards, cultures Optical Density at 600nm (OD600) was measured with a spectrophotometer, and readjusted to the desired OD by diluting with agroinfiltration solution. If coinfiltration with different genetic constructions is needed, A.tumefaciens culture with the corresponding plasmids were equally mixed.

Agroinfiltration mix was inoculated in 4 weeks old N. benthamiana plants using needleless syringes through abaxial surface of the three youngest leaves of each plant. Plant growing conditions were 16 light hours at 24ºC and 8 dark hours photoperiod at 20ºC. Finally, leaves samples were taken 5 days post infiltration (d.p.i.).

**Valencia UPV iGEM** Team

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# Team:Valencia UPV/Results

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* TEAM
  + [Team](https://2017.igem.org/Team%3AValencia_UPV/Team)
  + [Attributions](https://2017.igem.org/Team%3AValencia_UPV/Attributions)
* PROJECT
  + [Description](https://2017.igem.org/Team%3AValencia_UPV/Description)
  + [Results](https://2017.igem.org/Team%3AValencia_UPV/Results)
  + [Applied Design](https://2017.igem.org/Team%3AValencia_UPV/Applied_Design)
  + [Collaborations](https://2017.igem.org/Team%3AValencia_UPV/Collaborations)
  + [Achievements](https://2017.igem.org/Team%3AValencia_UPV/Achievement)
  + [Safety](https://2017.igem.org/Team%3AValencia_UPV/Safety)
* WET LAB
  + [Biological Design](https://2017.igem.org/Team%3AValencia_UPV/Design)
  + [Experiments](https://2017.igem.org/Team%3AValencia_UPV/Experiments)
  + [Protocols](https://2017.igem.org/Team%3AValencia_UPV/Protocols)
  + [Notebook](https://2017.igem.org/Team%3AValencia_UPV/Notebook)
  + [Contribution](https://2017.igem.org/Team%3AValencia_UPV/Contribution)
  + [Improve](https://2017.igem.org/Team%3AValencia_UPV/Improve)
  + [PlantSynBio](https://2017.igem.org/Team%3AValencia_UPV/Plant)
  + [Parts](https://2017.igem.org/Team%3AValencia_UPV/Parts)
* DRY LAB
  + [Modeling](https://2017.igem.org/Team%3AValencia_UPV/Model)
  + [Software](https://2017.igem.org/Team%3AValencia_UPV/Software)
  + [Hardware](https://2017.igem.org/Team%3AValencia_UPV/Hardware)
  + [Graphic Design](https://2017.igem.org/Team%3AValencia_UPV/GraphicDesign)
* HUMAN PRACTICES
  + [Outreach](https://2017.igem.org/Team%3AValencia_UPV/HP/Silver)
  + [Integrated and gold](https://2017.igem.org/Team%3AValencia_UPV/HP/Gold_Integrated)

Home • Wet Lab •

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### RESULTS

[PDF](https://static.igem.org/mediawiki/2017/f/f1/Valencia_UPV_Results.pdf)

## ChatterPlant’s results

Aiming to establish a bidirectional communication with plants, two different channels for interchanging information with them were developed (these channels are addressed in modules 1 and 2). Furthermore, seeking the goal of obtaining a future sustainable agriculture, a hardware device was designed and built (module 3) and a software tool that facilitates Plant SynBio knowledge was developed (module 4).

* **Module 1**: An optogenetic toggle switch circuit capable of tuning the expression of a desired protein depending on the wavelength of the light stimuli was developed.
  + Red activation of the optogenetic toggle switch was characterized.
  + Root-aerial part transport by viral autoreplicative vectors was demonstrated.
* **Module 2**: A Color Code System triggered by external inducible stimulus together with stress-signals was achieved in order to flag any change in their status accelerating corrective measures.
  + PhiC31 was characterized.
  + Metabolic load vs unproductive complex theory hypothesis were assessed.
  + Recombination Directionality Factor (RDF) action was demonstrated.
  + RDF was characterized.
  + Stress-inducible promoter feasibility was demonstrated.
  + Color code protein expression through a viral vector strategy was achieved.
  + Chromoproteins expression in *Nicotiana benthamiana* was tested.
* **Module 3:** A hardware device (ChatterBox) was engineered in order to provide a SynBio-based solution to improve agriculture sustainability.
* **Module 4:** PlantLabCo, an open-access platform aiming to unify Plant SynBio researchers work, was developed.

## MODULE 1: Human – Plant communication channel

ChatterPlant is a SynBio-based project that works as an interface between plants and humans to increase control over plant physiology. The human-plant communication channel comprises a root-specific modular optogenetic circuit that enables low-cost control on plants´ endogenous gene expression, therefore linking desired physiological outputs (e.g flowering) to precise, cheap environmental signals (light pulses), rather than expensive-to-create artificial environmental conditions.

Therefore, we demonstrated the performance of the light-inducible system based on red/far-red light in order to control the expression on any desired protein (Müller, Naumann, Weber & Zurbriggen, 2015).

#### CHARACTERIZATION OF RED/FAR RED GENETIC SWITCH

In order to test the red-light switch, a GoldenBraid multipartite assembly reaction was performed to create a genetic construct composed by the transcriptional factor, PIF6, fused to a DNA-binding domain (E) and the photoreceptor (PhyB) fused to the activator domain (VP64) together with a nuclear location sequence (NLS). When PhyB (which carry a VP64 activator domain fused to it) is irradiated with 660nm light, its conformation changes and it interacts with PIF6 at the promoter site sparking off the signalling pathway (Fig. 1). A luciferase gene regulated by a minCMV promoter containing an Etr8 operator site was used as a reporter gene in order to characterize the behaviour of this optogenetic circuit.

Following the [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Experiments#optogenetic), our data shows differential expression rates between the ON state (red light) and the OFF state (dark conditions). As Figure 1C and 1D shows, plants irradiated with red light showed an increasing luciferase activity after 23 hours from red-light activation, plants kept in dark conditions exhibited lower expression rates (corresponding with the basal expression) and plants irradiated with white light showed a varying behaviour response. Constitutive expression controls under strong (35s promoter) and weak (pNOS promoter) promoters were treated in white light and dark conditions.

Note that in the graphs below same error bars have negative values. This is due to the assumption of symetrical standard error when Matlab is computing error bars. In this case, since there is a small sample size (n) and the standard deviation (s) has values close to the mean, the standard error (s/ sqrt(n)) is nearly the standard deviation, and consequently it is also close to the mean value. The positive value of the standard error is calculated, and because of the mentioned simetry assumption, the same absolute value is assumed for the negative standard error. Since Matlab does not have any non-negativity restriction imposed on this estimation, it is possible that the estimated standard error is higher than the mean value of the samples, obtaining negative results when the standard error is substracted to the mean.

![](https://static.igem.org/mediawiki/2017/e/ee/Result_1.jpeg)

## MODULE 2: Plant – Human communication channel

As far as communication is concerned, a bidirectional channel must be established. In our work, we demonstrate that not only delivering orders into plants is feasible but also that plants are able to notify us of any change in their status.

A leaf’s colour change triggered by a viral vector approach apprises us whether plants are affected by any stress through our Colour Code System. Thus, we resolved to control biotic and abiotic stresses using a modular and orthogonal genetic AND gate in order to decrease leakage possibilities.

#### CHARACTERIZATION OF PhiC31 RECOMBINASE ACTION

PhiC31 is a serine recombinase with the ability of performing site-specific recombination between two attachment sites attP and attB (Keravala et al., 2006). We demonstrate that PhiC31 allows an accurate regulation of luciferase expression when it performs the inversion between its attachment sites. Following this [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Experiments#Phic31promot) we were able to characterize recombination performance under weak and strong plant-specific promoters (Pnos and p35s respectively).

[Reporter assembly construct](https://2017.igem.org/Team%3AValencia_UPV/Improve) is composed by a 35s promoter sequence and Mtb terminator flanked by two opposing PhiC31 site-specific recombination sites (Figure 5).

![](https://static.igem.org/mediawiki/2017/a/a6/ContributionImage1.png)

***Figure 5.** Graphic representation of Plant-Human genetic circuit with the construct comprised by a promoter and a terminator in opposite directions flanked by ΦC31 attachment sites. a) In presence of PhiC31 integrase, recombination occurs allowing the expression of a translation of a nonsense sequence or stuffer fragment . In this case, the circuit is in OFF state b) Graphic representation of the toggle switch in ON state only when PhiC31 and gp3 are expressed. Only when the promoter is activated under stress conditions, corresponding color protein will be expressed.*

These results showed a counterintuitive correlation between the recombinase and luciferase. The higher the expression of recombinase is, the lower the probability that inversion occurs is. That is, the more concentration of recombinase in plant cells, the fewer inversion efficiency is.

![](https://static.igem.org/mediawiki/2017/6/63/ContributionImage2.png)

***Figure 6.** a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by PhiC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when PhiC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by PhiC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Genetic construct that allows constitutive expression of phiC31 in the plant under the control of a weak constitutive promoter. e) Transcriptional unit for the expression of the Firefly Luciferase under the control of a strong constitutive promoter. f) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter. g) Bars chart representing luciferase expression levels before and after induction with PhiC31.*

After analysing the data obtained from luciferase assay, it can be observed at Figure 4:

1) A significantly difference between the OFF and the ON state expression levels.

2) The recombinase expression under a weak promoter (pNOS promoter) shows the same luciferase expression that the constitutive positive control (genetic construct which is expressing constitutively luciferase protein under the strong promoter).

3) However, the recombinase expression under the strong promoter shows the same expression level of the constitutive weak control (genetic construct based on the constitutive expression of luciferase under a pNOS weak promoter)

The likelihood of producing the inversion event should be directly proportional to the luciferase protein concentration inside plant cell. Consequently, more quantity of phiC31 recombinase entails more probability of activating reporter gene expression. However, this experiment shows that once recombinase expression exceeds a certain threshold, the effect changes so the likelihood of up-regulating the reporter gene expression decreases.

In order to provide robust evidences to the hypothesis explained above, a subsequent [experiment](https://2017.igem.org/Team%3AValencia_UPV/Experiments#Phic31Per) was performed. In this case, PhiC31 behaviour was analysed while being regulated by a weak promoter (pNOS) together with a optical density (*A.tumefaciens* culture concentration) was 10-fold higher than the used in aforementioned experiment (Figure 7).

![](https://static.igem.org/mediawiki/2017/7/74/ContributionImage3.png)

***Figure 7.** a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by PhiC31 attachment sites (attB and attP). It represents the negative control of our experiment. Only when PhiC31 inversion occurs, luciferase protein will be expressed. b) Genetic construct that allows constitutive expression of PhiC31 in the plant under a strong promoter c) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by PhiC31 recombined attachment sites (attR and attL). In normal basis, the promoter is inverted, allowing the expression of luciferase protein. It represents the positive control of our experiment. d) Plot representing PhiC31 behavior when infiltrated at OD 0,1.*

**Conclusion:** It was demonstrated the proper PhiC31 functioning and determined its behavior under different protein concentrations and promoter strengths. Although the expected hypothesis may be that the more recombinase concentration the more inversion events,the obtained results suggest the existence of a threshold above which that assumption is not true. Therefore, a low recombinase expression is needed in order to maximize recombinase action.

Therefore, the following question raised: **What is the reason for this counter-intuitive phenomenon?** We proposed two hypotheses which may explain this:

1. The high expression of recombinase may demand too much cell resources, causing a metabolic overload. Thus, luciferase expression decreases due to resource competition.
2. A PhiC31 tetramer binds to the attachment sites and it triggers the recombinase action. However, when PhiC31 is overexpressed, unproductive complexes are formed in the site-specific recombination sites obstructing the inversion event.

#### METABOLIC LOAD VS UNPRODUCTIVE COMPLEX HYPOTHESIS

An experimental approach was performed in order to demonstrate if the metabolic load hypothesis is correct. Following the [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Experiments#metabolic) it was proved that there is no relation between a higher expression of PhiC31 with a lower luciferase expression in the plant cell (Figure 8).

![](https://static.igem.org/mediawiki/2017/d/d5/Results_8.jpeg)

***Figure 8.** a) Graphic representation of the construct comprised by a promoter and a terminator in opposite directions flanked by PhiC31 recombined attachment sites (OD\* 0,02). b) Genetic construct that allows constitutive expression of phiC31 under a weak promoter. It was tested at several concentrations (OD\* 0,01 and OD\* 0,02). c) Construct that allows constitutive expression of luciferase under a strong promoter (35s Promoter). d) Graphic representation of luciferase expression levels after 48 hours post infiltration (blue) and 96 hours post infiltration (red). \*Final Optical Density of Agrobacterium tumefaciens culture.*

As Figure 8 shows, the expression of luciferase under the strong promoter (35s promoter) decreases along with time because of the high metabolic load in the cell plant. However, there is no differential luciferase expression between both different recombinase expression concentrations when the inducible reporter was already activated before adding PhiC31. Suggesting that decrease of luciferase expression observed in the aforementioned experiment was not caused by metabolic load but by the formation of PhiC31 unproductive complexes.

**Conclusion**: We demonstrate that metabolic load cannot explain the counter-intuitive hypothesis previously reported. That means PhiC31 unproductive complex formation hypothesis may be the correct one.

#### RECOMBINASE DIRECTIONALITY FACTOR (RDF) PERFORMANCE

In order to increase the genetic control over the expression of any color inducing protein through a viral vector strategy, a modular and orthogonal AND gate was designed. In normal basis, plant would be constitutively expressing PhiC31 recombinase and the stress-inducible promoter would be in OFF state (i.e. in opposite direction to the desired CDS) . In this case, PhiC31 attachment sites have been already recombined (attR and attL), being PhiC31 uncapable of inversing them back by itself. To do so a helper protein is required, so called recombinase directionality factor ([RDF](https://2017.igem.org/Team%3AValencia_UPV/Design)). With this approach recombinase switch becomes more robust, stable and less leaky.

![](https://static.igem.org/mediawiki/2017/a/a6/Results_9.png)

***Figure 9. Dynamic performance between RDF and PhiC31.** a) Graphic representation of the register assembly comprised by a promoter and a terminator with the recombined sites. It represents the negative control in the graphic. b) Genetic construct that allows a constitutively expression of PhiC31 under a **weak** promoter (OD\* **0,01**) c) Graphic representation of the genetic construct comprised by a promoter and a terminator with the attachment sites. It represents the positive control in the graphic. d) Graphic representation of the gp3 genetic construct. Its expression is under a **strong** promoter and was tested in both treatments (OD\* **0,1** and OD\* **0,2**). \*Final Optical Density of Agrobacterium tumefaciens culture. e) Plot of the luciferase expression levels obtained over time. f) Bars chart representing the difference of luciferase expression levels obtained before and after RDF induction.*

Recombination directionality factor’s (RDF or gp3) ability to reverse the integrase actuation direction allowing the inducible stress-promoter to acquire the active state was demonstrated in this experiment. Following the [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Experiments#GP3) we proved the effectiveness of the RDF in the recombination system.

As can be seen in Figure 9, the expression of gp3 together with PhiC31 trigger luciferase expression obtaining expression levels similars to a weak promoter (Pnos promoter). Interestingly, the highest level of luciferase expression (expression levels similar to a weak promoter — pNOS) was obtained with a low RDF expression (infiltrated at OD 0.1). Whereas, when the RDF was relatively high (infiltrated at OD 0.1, that is 2-fold in comparison with the other experimental treatment), the activation was lower than a weak promoter (pNOS) expression levels.

**Conclusion:** The ability of the recombinase directionality factor (RDF) to reverse the integrase action direction was demonstrated. Thus, postulating as the best way to control the direction of the stress-inducible promoter to express a color inducing protein.

#### RDF CHARACTERIZATION

RDF:phiC31 ratios define re-inversion efficiency. Bearing that in mind, we resolved to demonstrate that the optimal ratio between RDF and PhiC31 is 20:1 using a weak promoter for the recombinase expression and a strong one for the RDF. However, several experiments were performanced in order to demonstrate this hypothesis.

![](https://static.igem.org/mediawiki/2017/1/18/Results_10.jpeg)

***Figure 10. Dynamic performance between RDF and PhiC31.*** *a) Graphic representation of the register assembly comprised by a promoter and a terminator with the recombined sites. It represents the negative control in the graphic. b) Genetic construct that allows a constitutively expression of PhiC31 under a* ***strong*** *promoter (OD\** ***0,1****) c) Graphic representation of the genetic construct comprised by a promoter and a terminator with the attachment sites. It represents the positive control in the graphic. d) Graphic representation of the gp3 genetic construct. Its expression is under a* ***strong*** *promoter and was tested in both treatments (OD\** ***0,05*** *and OD\** ***0,15****). e) Line graph representing the data obtained of the level of luciferase expression over time for RDF module infiltrated at OD 0.05 and 0.15. f) Line graph representing the data obtained of the level of luciferase expression over time for RDF module infiltrated at OD 0.2 and 0.35. \*Final Optical Density of Agrobacterium tumefaciens culture.*

As can be seen in Figure 10 e) and 10 f), any of the RDF’s ODs assessed were able to trigger luciferase expression (i.e. re-inverting the promoter).

**Conclusion:** These results help to corroborate the unproductive complex hypothesis. When PhiC31 expression is under a strong promoter regulation, the formation of this complexes impedes the site-specific recombination event.

#### STRESS- INDUCIBLE PROMOTER

ChatterPlant proposes a modular and orthogonal [AND gate](https://2017.igem.org/Team%3AValencia_UPV/Design) as regulatory system for the color inducing protein. In this circuit leaf’s color changes are triggered by the designed genetic toggle switch comprised by an invertible stress-inducible promoter. This promoter up-regulates the expression of tobacco pathogenesis-related protein 1a ([Pr-1a](https://2017.igem.org/Team%3AValencia_UPV/Parts#basicPartsPH)) in presence of salicylic acid (pathogenesis signaling phytohormone).

Pr-1a promoter feasibility inside our circuit for flagging pathogen presence was assesed following the [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Experiments#stress).

![](https://static.igem.org/mediawiki/2017/a/a0/Results_11.png)

***Figure 11.*** *a) Transcriptional unit for the expression of the Firefly luciferase under the control of a stress promoter. b) Transcriptional unit for the expression of the Firefly luciferase under the control of the stress promoter when Salicylic acid is being synthesized c) Transcriptional unit for the expression of the Firefly luciferase under the control of a weak promoter. d) Graphic representation of ratios luciferase/renilla of all treatments explained previously.*

As can be seen in Figure 11, a significative differential expression were obtained between the salicylic acid treatment and the mock one (just water). That means, those plants infiltrated with the inducible construct and treated with salicylic acid presented higher levels of luciferase expression over those treated only with water.

**Conclusion**: Results show the suitability of using Pr1-a promoter for up-regulating expression levels. Therefore, Pr1-a and potentially other types of inducible promoters, postulate as candidates to develop a stress-inducible color code system.

#### COLOR CODE PROTEIN EXPRESSION BY VIRAL VECTOR DELIVERY

Viral vector auto-replicative capacity and cell-to-cell movement convert this strategy into one of the best strategies for high protein expression and yield production. Thus, we developed our proof of concept for this approach using the Tobacco etch virus (TEV) as a vehicle for the transport of the color inducing protein Rosea 1 (Ros1). This protein, Rosea 1 is an Antirrhinum majus transcription factor and it has been reported as an ideal visual reporter gene. Following the [experimental design](https://2017.igem.org/Team%3AValencia_UPV/Design), different N.benthamiana plants were infiltrated with A.tumefaciens culture carrying a TEV vector with Ros1 insert.

![](https://static.igem.org/mediawiki/2017/0/02/Results_12.png)

***Figure 12**. Images obtained two weeks after infiltrating A.tumefaciens carrying a PVX vector with a Ros1 insert inside it.*

**Conclusion:** As shown in Figure 12, the suitability of plant viral vectors as color inducing system vehicles was demonstrated. Furthermore, the viral vector’s systemic movement was proved since the agroinfiltration was performed in old leaves while color change was observed not in the old leaves but in the younger ones.

#### CHROMOPROTEINS

Chromoproteins suppose a simple and effective way of inducing different color changes in a desired organism. Its implementation in our circuit, would confer it with versatility, facilitating the creation of a color code system. However, to date, a color change in plant organisms has not been reported.

In order to expand our Color Code System, we propose using chromoproteins since their absorbance spectrum is in the visible one and non-special equipment is necessary. Amil CP, AmajLime and eforRed (BioBricks supplied to the Registry by Uppsala iGEM team between 2011 and 2013) were codon-optimized for Nicotiana benthamiana in order to make its expression easier.

**Conclusion:** Unfortunately, no color change was observed after infiltrating A.tumefaciens culture with the different chromoproteins (AmilCP, AmajLime and eforRed) either with their E.coli codon optimized sequence or with their plant codon optimized. Future experiments could be carried out after adding a chloroplast location sequence to these proteins. The transport of the chromoproteins to the chloroplast may help to their correct folding since its redox environment is more similar to the prokaryotic cytosol.

## MODULE 3: CHATTERBOX

Our aim with ChatterPlant was to increase sustainability of food production with a Synbio approach. To reach this final end different systems were tested and finally implemented in the device known as ChatterBox.

The first one is **temperature regulation**, key to control plant’s metabolism and homeostasis. With our refrigeration system we were able to stabilize 20 ºC in the growing chamber, an ideal temperature for growth.

![](https://static.igem.org/mediawiki/2017/8/85/Results_13.jpeg)

***Figure 13.** Graphic representation of the temperature control carried out by the ChatterBox system.*

Also, **humidity** is a very important factor to keep in mind, a low humidity level can induce stress, so we designed and tested a low cost DIY humidifier capable of proving a fine mist.

![](https://static.igem.org/mediawiki/2017/0/0c/Results_14.jpeg)

***Figure 14**. Picture of a ChatterBox prototype used in one of our first light-induction expression experiments.*

Next step, light. With the white LED strips direct sunlight was substituted. Entering the SynBio field, with the “**Optogenetic lamps**” and their specific wavelength the conformation of a protein was changed in the plant’s genome.

Finally, **color changes** in the leaves caused by an infection were detected with the camera and recognition software as shown in Figure 15

![](https://static.igem.org/mediawiki/2017/5/5e/Captura-1129-.PNG)

***Figure 15.** Demonstration of the results of the visual recognition software (right) after processing a real plant picture with induction of leave’s color change (left).*

## MODULE 4: PLANTLABCO

![](https://static.igem.org/mediawiki/2017/5/51/Results_16.png)

***Figure 16**. Frontpage of PlantLabCo online platform.*

The objective of PlantLabCo was to provide a solution to the main problems that Plant SynBio researches are facing up to know. In order to achieve this goal we studied the different tools that were provided in other fields of study to solve similar problems. After the proper research was taken we arrived to the point in which PlantLabCo is today. The four main approaches that we took were:

* Creating a forum where Plant SynBio researches can actively discuss and interact freely, discuss and explore every aspect of this field.
* Provide a web repository for experiments to be upload and consulted following a standardized system of information and metadata.
* Presenting a modeling software tool capable of recreating a vast amount of circuits to ease and help with the mathematical modeling as it is a key component on Plant SynBio
* An informative magazine that works as a medium to spread and educate the general public about this field

**Valencia UPV iGEM** Team

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