Difference between revisions of "Team:Navarra BG/project"

Fig. 1 Working in a biological safety cabinet.

The objective of our first experiment was to genetically modify plants of the species Nicotiana benthamiana to make them produce our proteins of interest, which bonded to the plant’s starch so that they could later get purified easily. To achieve it, we first needed to modify bacteria from the species Agrobacterium tumefaciens by introducing in them a plasmid that contained our gene. This way, we were able to use the modified bacteria as a vector to insert the genes in the plants.

The first step was building the plasmid that was later introduced in the bacteria and that contained the DNA fragment that we wanted to bring inside the plant’s genome. This DNA fragment had the information to produce two different proteins: the GBSS (Granule-Bound Starch Synthase), that increases the production of starch and bonds to the starch; and the GFP (Green Fluorescent Protein), that gets attached to the GBSS and served as a model protein. We chose it because under UV light it is fluorescent, so it would be easy for us to observe if the transformation worked as we wanted, but any other protein could be use in its place.

Fig. 2 E.Z.N.A. Plasmid DNA Mini Kit I

In order to build our plasmid, we mixed all the parts of the insert with an empty vector and with some enzymes, and we left the mix react in the thermomixer.

The next step was to introduce the plasmid inside bacteria from the species Escherichia coli through an electroporation.

After some hours, we seeded the bacteria using the method of the seeding balls and in a medium that contained, apart from other substances, an antibiotic, kanamycin, so we could select only the acteria that had our plasmid.

After they grew, we observed that there were blue colonies, which indicated that they had the empty plasmid, and white colonies, which indicated that they had the correct plasmid, with the insert.

Fig. 3 Nicotiana benthamiana plants

We selected the 6 best colonies, we put each of them in a separated recipient with LB, and we left them in an agitator until the next day.

When we got the recipients out if the agitator we centrifuged the liquid to separate the medium from the bacteria, and afterwards make the bacteria undergo a miniprep (Fig. 2). After purifying them, we kept the plasmids in a freezer.

We also seeded our plants of Nicotiana benthamiana.

After isolating and purifying our plasmids, we had to check that they had been correctly formed. To do this, we performed a digestion with restriction enzymes and an electrophoresis in an agarose gel. Only one of the samples we tried was positive, so we continued working with it.

Fig. 4 Agarose gel electrophoresis technique. Positive colony: 4

The next step was to build the definitive plasmid that we will put later in the plant. The plasmid we had already purified contained the genetic sequence we wanted the plant to have in the future, but because of its structure, it was not suitable to be introduced into the plant. So we did another reaction to take the transcriptional unit from our plasmid and a kanamycin resistance gene (something we would use in the future as a selection marker if we wanted to transform the plant stably), and introduce it all into a larger plasmid, suitable for the next steps. This is something that the cloning system we used, GoldenBraid, allowed us to make.

After that, we repeated what we had done with the other plasmid. We electroporated it in E. coli bacteria to clone it.

We also sterilized seeds of the plant Arabidopsis thaliana, sowed them in plates and left them growing in the growing chamber until next week, for a second experiment.

Once we had our final plasmid, the next step was to clone it once again. After introducing it in E. coli and then growing them in agar plates with spectinomycin, we picked the best white colonies (confirming the plasmid had been correctly added) and grow them in liquid LB at 37ºC.

The following step was to perform a miniprep to extract the cloned plasmids from our grown E. Coli. As we needed to confirm if the plasmid was correctly built we carried out an electrophoresis, with a faultless result:

Fig. 5 Agarose gel electrophoresis technique. Positive colonies.

The last step was to insert our correctly built plasmid in Agrobacterium tumefaciens (the bacteria that will finally be able to genetically modify our plants) conducting a simple lectrophoresis, and leave them grow in plates with rifampycin (to avoid the growth of other bacteria species) and spectinomycin, to finally be able to select the right colonies.

Subsequently, we transplanted the previously raised Arabidopsis thaliana to new plates, in groups of 6.

Fig. 6 Infiltration in the leaves of Nicotiana benthamiana.

We were then supposed to have our Agrobacterium tumefaciens bacteria with our well built plasmid, but we did a PCR and a gel electrophoresis to be sure.

Once we were sure everything was correct, we let bacteria grow in LB with rifampicin and spectinomycin at the same time we grew Agrobacterium tumefaciens with the silencing vector P19. For the infiltration, we mixed both liquids with the bacteria.

It was finally time to transform our plants (Fig. 6) The modification we did was transitory to confirm our method works, but in the future a stable modification will be possible.

We infiltrated our bacteria inside the plants leaves. We used a syringe and making a light pressure in the back part of the leaves the liquid got into them through the stomas. Inside the pant, the bacteria introduced our genes inside the plant’s cells.

We also started the second part of our experiment, which consisted on comparing how plants grew by their own or next to some microorganisms, like the fungus Alternaria alternata. It is known that some microorganisms like our fungus produce volatile compounds, that in contact with plants produce metabolism changes, which led to an increase in the starch production and the growing rate (Sanchez López et al. 2016). We used this to increase the starch production in our plants, and as a result, an increase in the amount of our protein we could produce.

The last step was to insert our correctly built plasmid in Agrobacterium tumefaciens (the bacteria that will finally be able to genetically modify our plants) conducting a simple lectrophoresis, and leave them grow in plates with rifampycin (to avoid the growth of other bacteria species) and spectinomycin, to finally be able to select the right colonies.

Subsequently, we transplanted the previously raised Arabidopsis thaliana to new plates, in groups of 6.

Once we had Arabidopsis thaliana wild type and fungus Alternaria alternata grown, we put some plants with the fungus and others without it and let them grow.

Fig. 7 Arabidopsis thaliana in contact with thefungus Alternaria alternata.

Our plants of Nicotiana benthamiana have been infiltrated by the bacteria Agrobacterium tumefaciens. Therefore, our plants were expressing already (producing) our target protein (GFP, Green Fluorescent Protein). Nevertheless, before continuing, we had to verify that our plants had really been modified. And for it, we observed some of the leaves with the help of a confocal microscope, which allowed us to see the fluorescence of the protein.

Fig. 8 Confocal microscope. Green color demonstrates
the presence of GFP in the chloroplast.

Fig. 9 Confocal microscope.

Once we knew for sure that the plants were producing GFP, the following step was to purify the protein. As we have already commented, an important part of our project is to manage to purify the target proteins (in our case, the GFP) in a simpler and cheap way with regard to the conventional methods. Developing our method, we think that we will make viable for astronauts the production of proteins of therapeutic and nutritional interest in the space.

Fig. 10 Microscope image where we can
observe the starch granules
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Our idea to simplify and cheapen the process of purification is to take advantage of the physical properties of a substance that plants produce in a natural form: starch. Starch is a compound that plants use to store the energy which they produce across the process of photosynthesis. In the process of its production many different enzymes operate. And one of them is the GBSS, Granule-Bound Starch Synthase, which will be very useful in our project because of its property of remaining stuck to the granules of starch. In addition, the starch is a very dense substance and is insoluble in water, which will be of help in the process of purification.

We have bound our protein to the GBSS, which will be bound to the starch granule. We think that, taking advantage of the density and insolubility in water of the starch, we can separate these joined substances (starch + GBSS + protein) through a simple centrifugation. Then, we will use a protease to break the binding between the GBSS and the target protein. The result we hope to achieve is a liquid with the highly purified protein, obtained in a fast, easy and cheap way.

Theoretically, all of the above should work, but experimental confirmation is needed to prove it. So we purified our protein. The first thing we did was to collect some leaves of our transformed plants and crush them. Then, we beated with a house blender them and filtered the liquid (Fig. 11) obtained to separate it from the solid remains of the leaves.

Fig. 11 Filtering the liquid for purification of the protein.

Next, we centrifuged the liquid to separate the starch + GBSS + GFP from the rest of the substances. When centrifuged, the starch precipitates and stays in the bottom part of the tube forming a pellet, while the rest of the liquid substances remain in the upper part forming the supernatant. We threw the supernatant and, voilà!: we already had our starch + GBSS + purified GFP. We take this photo of our starch under the microscope:

Fig. 12 Confocal microscope. Green color demonstrates the presence of GFP in the starch.

Next, we subjected the starch + GBSS + GFP to a lyophilization process, which serves to dehydrate it, so we can accurately measure the amount of starch we have been able to obtain from the leaves. Then we had to add the protease (in our case, we use factor Xa) that will cut the junction between the GBSS and our protein of interest (GFP). We also prepared a control with the sample without the protease, something that later will serve to show that it is really the protease what causes GFP to be released.

Finally, after the reaction, we only had to make one last centrifugation to separate the starch + GBSS, which remain in the bottom of the tube (pellet), of the GFP, which stay up (supernatant). With a pipette, we separated the supernatant from the pellet and passed it to another tube. And we already had our purified protein!

Regarding the other experiment (the one of volatile compounds emitted by microorganisms which affects plants) we got some results. We still have to quantify the difference in the amount of starch, but at this time, the difference in the growth of plants with fungus and plants without fungus is visible to the naked eye:

Fig. 13 Arabidopsis thaliana without (A) and in (B)
contact with the fungus Alternaria alternata.

We stayed for a few fantastic days in Barcelona taking part in the Spanish Meet-Up of the contest iGEM.

We modify our plants in order that they were producing our target protein, verify that they were doing it and purify this protein with our method. The last step of this experiment is to verify and to demonstrate if our method of purification had worked. That is to say, if we really had obtained the GFP (our protein).

Fig. 14 Cuvette used in the western blot technique.

To do it, we we use the technique Western Blot. The first step is to do an electroforesis. The idea is the same that in the electroforesis that we have done to separate the DNA, but this time we do it with proteins (Fig. 14). They go through a gel of acrilamida, that forms a kind of of microscopic network. After having applied an electrical current, the proteins begin to move across this network, and they had been separated according to their size (that allows them to move more or less easily) and their charge (that gives them more or less "force" to move).

Fig. 15 Developed in the western blot technique.

Once we have separated the proteins, the following step is to transfer them from the gel to a membrane. Later, we add a specific antibody for GFP, that is to say, a protein that adheres only to the GFP and no other protein. Later, we add the second antibody that adheres to the first antibody. This way, in our membrane we still have all the GFP molecules sticked to a protein which is sticked to a second protein. This second antibody has linked an enzyme that, in presence of specific substances, becomes visible. Because of it, to detect if it is or not GFP in the membrane, we only have to put it in contact with these substances and see if they appear stains. Where a stain appears, means that there is GFP.

As for the second experiment, now that we’ve obtained our target protein, we’ve focused on demonstrating how the volatile compounds emitted by certain microorganisms can make plants grow faster and produce greater amounts of starch. We want to increase the production of our target protein.

We cultivated two groups of plants from the species Arabidopsis thaliana: one of them in the presence of the fungi Alternaria alternata and the other one by its own, to compare both groups. Some days later, the difference in growth between the plants was very noticeable. What really mattered to us, however, was the amount of starch, so we measured it.

Fig. 16 Spectrophotometer for Multiskan microplates.

In order to do this, the first thing we did, harvest looks at the end of the light period, was extract as much starch as we could from the leaves, by crushing them with the aid of liquid nitrogen. Later, we separated this starch from other glucids, such as glucose or fructose, by using ethanol, heat and centrifugations.

After alkaline hydrolisis we quantified it. The first step to do so was to “break down” the starch in glucoses, using an amyloglucosidase enzyme. It was then ready to be measured using the spectrophotometer (Fig. 16) As expected, the results confirmed that the amounts of starch were much greater in the plants exposed to volatile compounds: from 10 to 12 times.