Team:Valencia UPV/Experiments

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Home • Wet Lab •

EXPERIMENTS

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INDEX

• Introduction
• 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
• Optogenetic 2.0
• GP3 Performance
• Metabolic Rate
• Chromoproteins
• Chromoproteins-PVX
• Stress-Promoter PR-1A

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

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 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

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

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

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

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

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

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

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

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

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

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