Bimolecular Fluorescence Complementation and Immunocytochemistry of Pap

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Materials and Methods:

The experiment was carried out as outlined in the lab manual provided through Moodle website [10]. The first part was to infiltrate the N. benthamiana with the Agrobacterium cultures. An initial OD600nm was taken, then the bacterial samples were pelleted for 10 minutes at 2000x g at room temperature. The cells were resuspended in the agroinfiltration solution to obtain an OD600nm of 0.5-0.6 and kept it for three hours at room temperature. The bacterial solutions in each combo were mixed in a 1:1:1 ratio. There were three sets experimental (combo 1 and 2), positive control (combo 5 and 6), and negative control (combo7). The following table lists the content of each combo and which table each combination was:

Table (1): different combos were assigned to each group in the lab to assess the viability of the proteins interaction and the presence of a complete YFP protein, this was done in day 01 of the experiment.

Combo 1 (experimental)

pGreen/ntGFP-PAP + pGreen/ctGFP-PAP +PTGS plasmid

Combo 2 (experimental)

pGreen/ntGFP-PAPx + pGreen/ctGFP-PAPx +PTGS plasmid

Combo 5 (positive control)

pGreen/ntGFP-4G + pGreen/ctGFP-4E + PTGS plasmid

Combo 6 (positive control)

pGreen/ctGFP-4G + pGreen/ntGFP-4E + PTGS plasmid

Combo 7 (negative control)

pGreen/ntGFP + pGreen/ctGFP +PTGS plasmid (p0/HCpro)

The Agrobacterium culture was administrated to the tobacco plants by slightly making an incision cut to the leaf where we can inject the bacterium culture. This was performed on two tobacco plants to as many as possible leaves. The plants then were left to grow for few days in the greenhouse to permit the proteins expression. On the second day of the experiment the leaves were sectioned to thin slides and placed in a fixative solution and left to fix for two hours. Each leaf was infiltrated with different Agro constructs and were placed in a plastic bag to prevent it from getting dry and to preserve the aldehyde fumes of the fixative solution. The leaves were washed in PEM for 15 minutes, then placed on a glass slides and mounted in a medium that contained the antifade agent. The slides were stored in a dark room until were examined by the confocal microscope. Pokeweed, toPAPcco (transgenic tobacco) plants were provided and each group picked one to perform the immunocytochemistry technique using them. The leaf of each plant was cut near the major vein and tissue were cut into 1 cm squares, which then were cut into smaller sections, and placed into Petri dish with the fixative solution for two hours. Sections then were washed in PEM for 15 minutes at a low speed and incubated overnight at 40C with anti-PAP polyclonal antibody (1:1500) in 3% BSA, 0.1% IGEPAL. After two days the sections were washed three times for five minutes in PBS, and incubated in the dark for two hours at room temperature with the secondary antibody. Again sections were washed with PBS for five minutes and were placed on a glass slides topped with antifade containing medium, and were stored in the dark until seen under the microscope. The materials used in this lab were mainly fixative solution, which contained 4% formaldehyde, 0.1% glutaraldehyde, 2.75% sucrose, 0.1% Ninidet P-40. PEM was in a ratio of 50 mM PIPES pH of 6.9, 5 mM EGTA, and 5 mM MgSO4. The Agrobacterium strains were AGL1, HCPro, P0, and P19.

Results:

The initial OD600 nm reading was taking for each tube containing pGreen by taking 0.5 ml from each of the tubes. We had 4 tubes contain different constructs as mentioned in table (2) below. We were left with 2.5 ml of each tube and we had to calculate the volume of the suspension solution to be added into each tube, which is also shown in table (2), and below is a sample of the calculation:

For tube 1 we had an OD600 nm = 1.329

X = the volume required

So 1.329 * 2 * 2.5 = 1 * X

X= 6.645 ml

Table (2): The results of five tubes containing different constructs that were prepared at the first day of the experiment to make the assigned combos combinations (2 and 6), and a control tube contains only the plasmid. The OD600 nm readings for each sample were taken and the suspension volume was calculated based on that reading to be added to the tubes to make the final volumes.

Tubes

Contents

OD600 nm Readings

Suspension volume

Tube 1

pGreen/ctGFP-4G

1.329

6.645 ml

Tube 2

pGreen/ctGFP-PAPx

0.783

3.915 ml

Tube 3

pGreen/ntGFP-4E

1.26

6.30 ml

Tube 4

pGreen/ntGFP-PAPx

0.537

2.685 ml

Tube 5

PTGS plasmid

1.025

5.125 ml

Table (3): The results of BiCF system where each combo observed under the confocal microscope, each group had two different set of combos. After the results were photographed using a built in camera they were shared among the class. The pictures were taking under different magnifications mostly it was under 30x magnification. The plants used was N. benthamiana infiltrated with Agrobacterium culture containing the PAP construct fused with fluorescent protein. Examination was at room temperature.

Combos numbers

Results

Combo