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EXPERIMENTS

INDEX

Phic31 Performance
Gp3 Performance-Reporter (L X R) - 1
Gp3 Performance-Reporter (L X R) - 2
Phic31 Performance (P X B)
Phic31 Performance-Promoters
Characterization Of Phic31 Performance
PVX
Optogenetic–Controlling the Genetic Expression with Light
GP3 Performance
Metabolic Rate
Chromoproteins
Chromoproteins-PVX
Stress-Promoter PR-1A

PHIC31 PERFORMANCE

Objective

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

Plant Chassis

Nicotiana benthamiana

Parts

Figure 1. 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, allowing the characterization of luciferase dynamic. 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 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 1a and Figure 1c) was 0,02 and the optical density of PhiC31 construct (Figure 1b) was 0,05. 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. 1a and 1b) were agroinfiltrated on 12th August at 12:00h. After 48h post-infiltration (14th August), positive and negative control were sampled and the recombinase was agroinfiltrated. After 36h, leaves were sampled every eight hours finishing on 18th August at 16:00h. At this point, positive and negative controls were also sampled. Overall, six points of samples were taken in the assay after recombinase were agroinfiltrated.

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

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

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

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

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 54h 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

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 1a and Figure 1c) was 0,02 and the optical density of PhiC31 construct (Figure 1b) 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

Figure 8. 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 8a). 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

Figure 7. 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 7a and 7c). 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.

GP3 PERFORMANCE

Objective

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

Plant Chassis

Nicotiana benthamiana

Parts

Figure 9. 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. 9a and c) were 0,01, the optical density of PhiC31 construct (F.9b) was 0,005 and the optical density of gp3 construct (F.9d) 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. 9a) was agroinfiltrated together with recombinase (Fig. 7c) on 12th October at 18:00h. Positive and negative controls (Fig. 9a and Fig. 9c) were infiltrated together with the internal controls (Fig. 9e and 9f). After 48h post-infiltration (14th October), the RDF (Fig. 9d) was agroinfiltrated letting a control plant without being agroinfiltrated. After 36h, leaves were sampled every eight 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 RATE

Objective

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

Plant Chassis

Nicotiana benthamiana

Parts

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 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 10a) was 0,02 and the optical density of PhiC31 construct (Figure 10b) 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. 10a) was agroinfiltrated together with the recombinase (Fig.10b) 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

Figure 11. 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. 11a 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

Figure 12. 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. 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.

STRESS-PROMOTER PR-1A

Objective

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

Plant Chassis

Nicotiana benthamiana

Parts

Figure 13. 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.

@@ Line 289: / Line 289: @@
 								<li class="list-group-item"><a href="#procedures2">Gp3 Performance-Reporter (L X R) - 2</a></li>
 								<li class="list-group-item"><a href="#Phic31Per">Phic31 Performance (P X B)</a></li>
-								<li class="list-group-item"><a href="#Phic31promot">Phic31 Performance Under Different Promoters</a></li>
+								<li class="list-group-item"><a href="#Phic31promot">Phic31 Performance-Promoters</a></li>
 								<li class="list-group-item"><a href="#Phic31carac">Characterization Of Phic31 Performance</a></li>
+								<li class="list-group-item"><a href="#PVX">PVX</a></li>
+								<li class="list-group-item"><a href="#optogenetic"></a>Optogenetic–Controlling the Genetic Expression with Light</li>
+								<li class="list-group-item"><a href="#GP3"></a>GP3 Performance</li>
+								<li class="list-group-item"><a href="#metabolic"></a>Metabolic Rate</li>
+								<li class="list-group-item"><a href="#chromoproteins"></a>Chromoproteins</li>
+								<li class="list-group-item"><a href="#chromoPVX"></a>Chromoproteins-PVX</li>
+								<li class="list-group-item"><a href="#stress"></a>Stress-Promoter PR-1A</li>
 							</ul>
 						</div>
@@ Line 305: / Line 312: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
-                                                                <img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/c/c4/Exp1.png"/>
+                                <img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/4/4c/Experimento1Valencia.png"/>
 								<p class="jopesangria"><i>Figure 1. 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, allowing the characterization of luciferase dynamic. 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 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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 322: / Line 329: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
- <img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/2/24/Exp231.png"/>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/b/b2/Experimento2_3Valencia.png"/>
 								<p class="jopesangria"><i>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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 339: / Line 346: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
- <img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/2/24/Exp231.png"/>
+								<img class="float-left img-fluid" style="width:100%" src="https://static.igem.org/mediawiki/2017/b/b2/Experimento2_3Valencia.png"/>
 								<p class="jopesangria"><i>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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 356: / Line 363: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/1/16/Experimento10Valencia.png"/>
 								<p class="jopesangria"><i>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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 365: / Line 373: @@
 						<div id="Phic31promot" class="container">
 							<div class="heading-color">
-								<h2><span>PHIC31 PERFORMANCE UNDER DIFFERENT PROMOTERS</span></h2>
+								<h2><span>PHIC31 PERFORMANCE - PROMOTERS</span></h2>
 							</div>
 								<h4><strong>Objective</strong></h4>
@@ Line 372: / Line 380: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/a/a4/Experimento5Valencia.png"/>
 								<p class="jopesangria"><i>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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 388: / Line 397: @@
 								<p>Nicotiana benthamiana</p>
 								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/3/38/Experimento6Valencia.png"/>
 								<p class="jopesangria"><i>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.</i></p>
 								<h4><strong>Method</strong></h4>
@@ Line 393: / Line 403: @@
 								<h4><strong>Timeline</strong></h4>
 								<p class="jopesangria">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.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="PVX" class="container">
+							<div class="heading-color">
+								<h2><span>PVX</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the viral systemic movement performed by Potato Virus X (PVX).</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/d/d4/Experimento7Valencia.png"/>
+								<p class="jopesangria"><i>Figure 8. a) Graphic representation of PVX-dsRED- Express Vector. When the viral vector is being produced, the fluorescent protein (dsRED) will be expressed.</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">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 8a). The final Optical Density of the genetic construct was 0,1.</a></u></p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">One week after transformation, plants were observed at the magnifier in order to check the fluorescence in the aerial part of the plant.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="optogenetic" class="container">
+							<div class="heading-color">
+								<h2><span>OPTOGENETIC – CONTROLLING THE GENETIC EXPRESSION WITH LIGHT</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">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.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/5/53/Experimento8Valencia.png"/>
+								<p class="jopesangria"><i>Figure 7. 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.</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> and subsequent <u><a data-toggle="modal" data-target="#luciferaseAssay">luciferase assay</a></u> 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 7a and 7c). 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.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">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.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="GP3" class="container">
+							<div class="heading-color">
+								<h2><span>GP3 PERFORMANCE</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the performance of gp3 using the register assembly construct with the recombined attachment sites LxR.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/a/a0/Experimento9Valencia.png"/>
+								<p class="jopesangria"><i>Figure 9. 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.</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> and subsequent <u><a data-toggle="modal" data-target="#luciferaseAssay">luciferase assay</a></u> were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (F. 9a and c) were 0,01, the optical density of PhiC31 construct (F.9b) was 0,005 and the optical density of gp3 construct (F.9d) 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.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">Register assembly construct (Fig. 9a) was agroinfiltrated together with recombinase (Fig. 7c) on 12th October at 18:00h. Positive and negative controls (Fig. 9a and Fig. 9c) were infiltrated together with the internal controls (Fig. 9e and 9f). After 48h post-infiltration (14th October), the RDF (Fig. 9d) was agroinfiltrated letting a control plant without being agroinfiltrated. After 36h, leaves were sampled every eight 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.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="metabolic" class="container">
+							<div class="heading-color">
+								<h2><span>METABOLIC RATE</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the metabolic rate of cells that can affect recombinase performance from the molecular to the organismal level.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/b/b7/Experimento4_10Valencia.png"/>
+								<p class="jopesangria"><i></i>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 luciferase protein. b) Genetic construct that allows constitutive expression of phiC31 in the plant under a weak promoter (Pnos promoter).</p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> and subsequent <u><a data-toggle="modal" data-target="#luciferaseAssay">luciferase assay</a></u> were performed in order to study gene expression at transcriptional level. The final Optical Density of reporter constructs (Figure 10a) was 0,02 and the optical density of PhiC31 construct (Figure 10b) 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.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">Register assembly construct (Fig. 10a) was agroinfiltrated together with the recombinase (Fig.10b) 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.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="chromoproteins" class="container">
+							<div class="heading-color">
+								<h2><span>CHROMOPROTEINS</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the performance chromoproteins in plants.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/5/5c/Experimento11Valencia.png"/>
+								<p class="jopesangria"><i>Figure 11. 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).</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> was performed in order to test chromoprotein gene expression. The final Optical Density of reporter constructs (F. 11a and b) were 0,1.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">One week after transformation, plants were observed at the magnifier in order to check the color protein in plant leaves.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="chromoPVX" class="container">
+							<div class="heading-color">
+								<h2><span>CHROMOPROTEINS-PVX</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the performance chromoproteins in plants.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/e/e5/Experimento12Valencia.png"/>
+								<p class="jopesangria"><i>Figure 12. 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.</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> was performed in order to test chromoprotein gene expression. The final Optical Density of reporter constructs (F. 12a and b) were 0,1.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">One week after transformation, plants were observed at the magnifier in order to check the color protein in plant leaves.</p>
+							<div class="divider"><!-- divider --></div>
+						</div>
+						<div id="stress" class="container">
+							<div class="heading-color">
+								<h2><span>STRESS-PROMOTER PR-1A</span></h2>
+							</div>
+								<h4><strong>Objective</strong></h4>
+								<p class="jopesangria">Testing the performance of the stress-promoter under several treatments.</p>
+								<h4><strong>Plant Chassis</strong></h4>
+								<p>Nicotiana benthamiana</p>
+								<h4><strong>Parts</strong></h4>
+								<img class="img-responsive" style="width:100%" src="https://static.igem.org/mediawiki/2017/1/1e/Experimento13Valencia.png"/>
+								<p class="jopesangria"><i>Figure 13. 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.</i></p>
+								<h4><strong>Method</strong></h4>
+								<p class="jopesangria">An <u><a data-toggle="modal" data-target="#agroinfiltration">agroinfiltration</a></u> and subsequent <u><a data-toggle="modal" data-target="#luciferaseAssay">luciferase assay</a></u> 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.</p>
+								<h4><strong>Timeline</strong></h4>
+								<p class="jopesangria">Genetic constructs were agroinfiltrated and after 70h post-infiltration, all plants were sampled.</p>
 							<div class="divider"><!-- divider --></div>
 						</div>