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Home • Wet Lab •

EXPERIMENTS

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INDEX

• Phic31 Performance
• Gp3 Performance-Reporter (L X R) - 1
• Gp3 Performance-Reporter (L X R) - 2
• Phic31 Performance (P X B)
• Phic31 Performance Under Different Promoters
• Characterization Of Phic31 Performance

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 UNDER DIFFERENT 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.

×

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