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

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<h3 class="about-title mb-70">First week</h3>
 
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<span class="pie"><strong>Fig. 1</strong> Working in a biological safety cabinet.</span>
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<p>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 <em>Agrobacterium tumefaciens</em> 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.</p>
 
<p>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 <em>Agrobacterium tumefaciens</em> 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.</p>
 
 
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<span class="pie"><strong>Fig. 1</strong> Working in a biological safety cabinet.</span>
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<p>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.</p>
 
<p>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.</p>
 
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Revision as of 07:49, 5 October 2018

The Project

What would you take to space? Imagine you where about to participate in an expedition to a planet far away from Earth and think in the things that you would take with you. Oxygen, water and food would be interesting things to begin with. Maybe some of you are also thinking of your favourite books, music, some electronic devices, something to write, to draw... You should also take medicines. Maybe you or another member of the crew needs them on a daily basis, or just in case anyone becomes ill during the expedition. Now, think: what if your trip was of 5, 10 or 15 years long? It may be possible to carry supplies for some months in the spaceship, but in those years everything will spoil. Its conservation would be hard and costly, and it would take too much space.

To avoid this, scientists from different parts of the world are working in making plants grow and survive in other habitats. Astronauts in the International Space Station have already eaten plants they had grown themselves in space. So, it looks like people participating in future long-term space missions would be able to grow and eat their own plants. However, how about nutrients that aren’t found naturally in plants? Or how about those medicines that we’ve recently talked about? How could astronauts produce specific protein compounds? Well, that is what our project is about.

Broadly speaking, large-scale production of recombinant proteins involves two steps: (i) synthesis of proteins using genetically modified organisms, and (ii) purification of the proteins. The first step has been mainly limited to Escherichia coli and Saccharomyces cerevisiae, although increasingly popular plant-based systems offer the potential for safe, economical and high-capacity production for many proteins of pharmaceutical and nutritional interest. Protein purification processes involve multiple steps. Various systems based on the production of genetically engineered fusion proteins (i.e. an affinity tag covalently linked to a target protein) have been developed to simplify these costly processes. Among them, the plant-based technology involving targeting of oleosin-fused proteins to organelles known as oilbodies has been shown to be cost- effective and enable high levels of production and purification of recombinant proteins.

Starch is the main storage carbohydrate in vascular plants, its abundance as a naturally occurring compound of living terrestrial biomass being surpassed only by cellulose. Synthesized by different isoforms of starch synthases (SS) this polyglucan accumulates as dense and insoluble granules in the plastids. One SS isoform (the GBSS) is bound to the starch granule. In this project we propose to develop a simple and cost- effective plant-based method for production and purification of recombinant proteins. The system is based on the production of plants transiently expressing a target protein (the green fluorescence protein, GFP) fused to GBSS. Transformed plant tissues will be milled in a suitable aqueous buffer and the starch granules will be purified from plant tissue-derived impurities through a series of simple centrifugation and wash/elution steps as in this aqueous environment the starch granule can be made to precipitate. The GBSS::GFP will be engineered to contain a unique cleavage site recognized by a specific protease, enabling the GFP to be separated from the GBSS into the aqueous buffer, while the GBSS remains embedded the starch granule. Once treated with the protease, the starch granules will be removed by centrifugation while the highly purified cleaved GFP can be further purified using conventional downstream processing.

First week

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.

Fig. 1 Working in a biological safety cabinet. Fig. 1 Working in a biological safety cabinet.

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 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 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.