This Is the End

THE End Has Come

                   Working in the laboratory and with the graduate students at the Biodesign Institute has taught me several key tools that I will never take for granted. I have learned more about biotechnology and its relationship with microbiology than I would ever had in a common classroom setting. I would like to thank the following graduate students: Richard, Ryan Jonathan, Ashley, and Cullen for all of their help in the laboratory. They have taught how it's like to be a college student and how working in a laboratory is a great way to showcase the knowledge that I have acquired from all of my years studying at BASIS. I would also want to thank my on-site mentor, Dr.Chen and Hualing, because they trusted me working in the laboratory and having my own portable office for me to work at.  These people have been great role models to me and showed how it is like to be a professional working in the laboratory in order to conduct research that has the potential of saving thousands of lives in the future. The research that I have acquired from my internship will benefit my future as I have obtained more knowledge and skills that I can apply this upcoming fall. Like I said before I have learned way better doing hands-on work than I would have learned from reading a book, and because of that I would like to say thank you to the entire Biodesign staff.

                     Another group that I have to thank are my counselors and my Basis mentor, Mrs. Sandor. Thank you for being there for me whenever I had questions about my research and figuring new ways to better organize the information that I have learned from the many articles that I have read over these last three months. The data tables that Mrs. Sandor has sent me are very helpful for this line of research and that I really appreciate having the chance to organize my results in a research model. Mrs. Sandor's comments and research support is something that I can never forget and I truly am thankful to have her as my mentor. So thank you again. Also, thank you Mrs. Q and Mrs. Kate for giving me this opportunity to study this line of research, because I have truly grown from this experience. I would like to thank you for keeping up with my blog posts and making sure that I have had all my assignments turned in. Again, thank you for allowing me to have a senior project in the first place, because I have truly appreciated the opportunities I have at the Biodesign Institute.

The Parameters to Plant Harvesting and Antibody Extraction

The Wonders to Plant Harvesting

                   Hello to all of my readers, today I would like to introduce to you all the last crucial method I have learned at the Biodesign Institute. Harvesting is a crucial method that I have used to extract the antibodies from the Nicotiana benthamiana plant leaves, which is the end result that I am working to obtain. A key point that I have to address to everyone is that harvesting involves two other techniques, which are clarification and protein A chromatography, that are important for the extraction of the antibodies from the leaves.

                Whenever I harvest a leaf, I have to first cut the leaf in half in order to remove the large, central stem vessel found on the leaf. This vessel and the plants' stems are useless to harvest, because these regions on the plants lack a suitable amount of antibodies for us to extract from; hence they have to be removed. Once a leaf is cut in half we have to remove any regions on the leaf that appear black or brownish, because these regions on a leaf indicate that there aren't a lot of antibodies that are produced. The reason why these leaves change color is either that the leaf didn't received enough water and fertilizer for it to grow healthy or it is a symptom of the leaf when it is infected by a plant bacterium, agrobacterium in this case. After all of the healthy, green leaf material has been cut off, I then have to put the leaf material into a blender in order to mash up the leaf material to form this green smoothie. The green smoothie is then filtered by using cheesecloth into four lab bottles, that are placed in a box of ice, so that the antibody proteins don't denature. What are we filtering, you may ask? We are trying to filter out the normal structural plant proteins that are found in the leaves such as rubisco for instance, so that all we have in the green smoothie are the antibodies. However, the cheesecloth isn't able to completely filter out all of the other plant proteins, enzymes, and chlorophyll pigments. That is where the two techniques, clarification and protein A chromatography, come into play.

                     The two techniques, clarification and protein A chromatography, that I mentioned above are used to extract the monoclonal antibodies from the smoothies. When I watch the graduate students perform the clarification technique, basically they continuously centrifuge the green smoothie until the green smoothie turns into a brown solution. The brown solution indicates that most of the leftover plant proteins and pigments are diminished in the smoothie, since the centripetal force produced from the centrifuge separates out these proteins and pigments from the antibodies. After the centrifuge does its job, that is when the graduate students are able to remove the separated pigments and plant proteins from the smoothie solution, which over time explains why the color of the smoothie changes color. After the graduate students finish clarification, we then move on to protein A chromatography. Protein A chromatography is basically the last filtering method that we will have to employ in order to extract the purified antibodies from this brown solution. The graduate students pour the brown solution into a lab column, where there is a filter attached and separates the divisions inside the column. Whatever solution that passes through this filter are the purified antibodies. We also have to inject a specialized affinity protein called protein A into the column. Using Protein A, which is a protein used in labs that has an affinity to an antibody's charge and attaches itself onto the antibody, the graduate students somehow were able to attach tiny iron beads to protein A. By attaching these iron beads to protein A the protein will not be able to attach to the antibodies, as these beads keep the protein A from passing through the filter. Now that this problem has been solved any solution that passes through the filter are the antibodies. The residue antibodies can then be pipetted into small lab tubes, where these tubes will hold the extracted antibodies that were able to be produced- by simple lab plants. These techniques are pretty much how we were able to extract the antibodies and show how important infiltrated plants really are in the laboratory. 

Agroinfiltration is More Complex Than I Originally Presumed

The Complexity of Agroinfiltration

             Good afternoon everyone, during today's internship experience I have discovered that one of my past blogs has some incorrect information about agroinfiltration. It turns out that agroinfiltration is more complex than I have originally presume. For instance, in the laboratory I have found that there are four different strains of agrobacterium media that are used to transport the gene of interest to the Nicotiana benthamiana plants. These different agrobacterium strains act as vectors, where like I said before they carry the gene of interest to the plants in order to express the genes and use the genetic sequence encoded by the gene of interest for the plant machinery to produce the desired monoclonal antibodies. In the laboratory, we have engineered the four different strains to possess plasmid genes from different vector sources. For instance, one of the agrobacterium strain possesses PVX (Potato Virus X) plasmid genes, another agrobacterium strain consists TMV (Tobacco Mosaic Virus) plasmid genes, another consists a genetically engineered heavy chain combined TMV plasmid genes, and lastly there is one strain that has a light chained combined PVX plasmid genes. A quick note that I think my audience should know is that a plasmid is a circular loop of DNA genes that is found in the nucleoid region of a bacterium that can replicate independently just like regular DNA chromosomes. Pretty much using plasmids has been the pivotal technique that is used in my project because these plasmids are responsible for containing the gene of interest that I need to infiltrate into the plants in order to trick the plants to produce the monoclonal antibodies. The heavy chain and light chain components are what we want to be used to replaced the normal viral proteins that are naturally found on PVX and TMV, where these chains are responsible for the bacteria to transmit the desired gene of interest into the plants. Pretty much these new facts about my project I felt had to be readdressed in my blogs, because I want anyone who is reading my blogs to understand the work that I doing in the lab. 

                 The production of the monoclonal antibody is pivotal to my project, because these antibodies serve as temporary treatments for patients infected with the Dengue virus. In one of my previous blogs, I have described the symptoms that are caused by the Dengue virus and that these symptoms progress over time. That is why it is very important to produce these antibodies in order for the antibodies to help opsonize the Dengue virus infection and prolong its effects onto an infected host until the host's immune system can kick in and destroy the threat. By no coincidence does the antibodies treat the host, because like any other antibody each of these antibodies are specific for one viral epitope. Key note: an epitope is a specific region on a viral envelope where an antibody can attach to a receptor on the viral surface. This treatment of the production of monoclonal antibodies is only temporary and may not work eventually as viruses such as Dengue continue to evolve and change the structure of their epitopes. It is a continued fight in the microbiology world against these pathogens where as long as the pathogens; particularly viruses, continue to evolve over time no one treatment will continue to work. It is a continuous fight where us, researchers, will have to find new ways to deal with the evolution of these pathogens or else our species and other species too will pay the price. Wish our species good luck in the future, because evolution of bacteria and viruses can cost the lives of any species.

As you can see, here is how monoclonal antibodies can be produced in a mammalian host.

Here is a picture demonstrating one example of viruses evolving

  

How Autoclaving Applies to Seed Pellets?

Why Autoclave Seed Pellets?

               Hello everyone, this is Armando again. I just wanted to let you all know that autoclaving has several more uses than I have explained in my previous blogs. Not only does autoclaving help kill any viral or bacterial pathogens that could reside on the surface of lab materials for instance, but also it turns out these pathogens can all be anchored onto the lab plants too. In the lab, when I prepare to transplant the plants into the different trays and separate out the wild type plants from the transgenic plants. I have to also take into consideration of any bacteria that could be living on the seed pellets, where these pellets are what hold together the soil and the plant seeds together. Below is a picture of several seed pellets:

                In order to autoclave the seed pellets what I had to do was first place all of the seed pellets into these large compartment trays not like the ones shown below. When I was doing this, there was about 60 pellets that could fit into each of the trays. Second, which is the easiest step, I simply added water into the trays so that by the time the pellets are placed into the autoclave machines the pellets should start to expand out. Third, I had to put the trays into plastic bio hazardous bags in order to indicate to other lab workers to be cautious around the trays and removing them from the autoclave machine. When I place the seed pellet trays into the autoclave machine, I had to set the mode of the autoclave machine into its liquid mode and gravity mode, because I am autoclaving a wet, bio hazardous material. The gravity mode is meant to for the autoclave machine to set its heat at a stable temperature to which the trays can't be melted at. While the liquid mode is to indicate to the machine to vaporize the water stored in the trays by converting the liquid water into a gas by adding excess heat into the oven. The gas will vaporize and exit through an air duct inside of the machine and thankfully the machine somehow contains the gas so that no bacteria can escape into the outside facility. Lastly, I have to play the wait game as I have to wait patiently for a whole hour till the pellets have been autoclaved. By the time the pellets are finish, I notice that the seed pellets have expanded to a point that the seed roots will easily be able to expand out in the soil and grow healthy, since there are no plant bacteria that can harm the plants. Now that is pretty much it to autoclaving seed pellets. It is a pretty simple, but repetitive process, but hey at least now the plants can grow healthy since any plant-infecting bacteria should be killed off by the heat produced from the autoclaved.

Here is a picture of wild type seed pellets.

I am waiting for the plants to bloom out from the seeds.

Results From the Laboratory

To Think How Much Work Was Achieved

                   Last week while working in the laboratory; I received news about the data collected for the amount of viable antibodies that were produced from the infiltrated plants. It turned out that for about each gram of leave material that was infiltrated around 350 mg of antibodies were produced! So far as I know the mass of the leaves is uncertain because in the laboratory I am dealing with several different sizes of leaves, making it difficult to figure out exactly an average mass for these plants' leaves. Typically the mass of the leaves I have dealt with can be around 5 to 7g of leave matter, but again the amount of leaf matter that is still available after infiltration can change.This number is super tremendous because thinking about it instead of injecting a common vaccination into a patient such as say giving the patient a subunit vaccination, which is injecting the pathogenic antigen into a patient's body, doctors can turn to injecting human-like antibodies into the patient. Think about it imagine injecting several hundreds of mg of antibodies into a host, where those antibodies can be used to combat the replication of a pathogen found inside a host without the need for the host's immune system to respond to the infection directly. Typically in most individuals the rate at which a human immune system produces its peak amount of antibodies that can respond to a specific strain of a virus is typically two weeks. Two weeks is a long time and by that time the infection could have gotten worse as the  virus mutates inside the human immune system, causing the virus' shape and antigen structure to change with it. Not only that but the usage of antibiotics to destroy the virus wouldn't work too because antibiotics can only target against bacteria and not viruses. The implementation of plant-based biological systems has greatly posed a better future for vaccination alternatives.

                  Although I have brought up some pros to the implementation of the plant-produced antibodies, there are still cons to this alternative that have to be addressed in the given future in order for this system to be efficient for a greater tested population. One obvious con that I hope everyone reading this asks to themselves is how can there be enough antibodies produced to supply for a larger population. Typically the production of these antibodies can take around 2 to 4 months, because the researchers have to grow the Nicotiana benthamiana plants and control their growth. In that case, there has to be thousands of plants to be produced and grown in the right conditions in order for there to be enough antibodies to be produced from these lab organisms. Not only the amount of time it takes for the plants to be produced but the decision to continue using Agrobacterium, PVX (Potato virus X), and TMV (Tobacco Mosaic virus) comes into play, because there has to be enough of these organisms to be grown to transmit the correct gene sequences for the plants to use to produce the antibodies. Also, there is a problem with using antibodies to defend a human body, because the antibodies are specific to one type of viral structure (active site) and so if the virus was to mutate then there is no way for the introduced antibodies to even work. Typically when making the antibodies there has to be enough buffer solution such as MES to be able to help stimulate growth of the bacteria, so that task right there can be a problem. The timing of this entire process and the issue that adaptive immunity isn't stimulated in a human immune system can be difficult potentials to the use of plant-made antibodies. It's better for immunity to be given to the human hosts instead of giving the patient antibodies by injecting a vaccination, but there is always an issue with viruses being able to change their structure due to mutations. This ability that viruses have is something that has to be addressed in order for any of these two mechanisms to work efficiently for the future populations that are exposed to viruses such as Dengue.

Here is a picture of the two types of adaptive immunity and the type of immune cells utilized in each type

              

Glycosylation and its Applications to the Transgenic Plants

Glycosylation and the Transgenic Plants

                 What is glycosylation, you may ask? Is it a process, a part of a plant, or even an artificial machine? No, but rather it's a mechanism that is performed by plants that is very crucial to understanding my project. Glycosylation is an enzymatic, site-specific process that takes place around the endoplasmic reticulum where a carbohydrate molecule is added to a target protein. In this sense, the carbohydrate helps to stabilize a protein's structure, allowing the protein to fold correctly. Another purpose for glycosylation is that it helps immune cells to be able to recognize host cells by developing sugar-binding proteins called lectins. These lectin proteins are what allow the immune cells to recognize other host cells by recognizing specific carbohydrate molecules, that are found on the surface of all host cells. As you can see clearly, this property is very important for a healthy immune to function property and not end up becoming an autoimmune disease.There are five different classes of glycosylation and they include: N-linked glycosylation, O-linked glycosylation, phosphor-linked glycosylation, c-linked glycosylation, and glypiation. For the development of the monoclonal antibodies at the Biodesign Institute, the researchers utilize N-linked glycosylation to change the structure of the Fab region of the antibodies. The Fab region of an antibody is the portion of the antibody that locks or hooks onto a specific cell surface for which in the case of antibodies is what allows the antibodies to opsonize a particular pathogen.     

Here are the three regions that make up an antibody.

             Hence, as you can see by changing the structure of the antibodies the researchers at the Biodesign Institute are altering the role of these proteins so that they can opsonize the specific Dengue virus. If I haven't mentioned it before, what I mean by opsonization is that it's an immune defense process where antibodies specific to a particular virus can surround the entire virus surface and hook onto the virus' surface receptors, keeping the virus from being able to infect a host cell and replicating. This ability to alter the structure of an antibody is very crucial to my project and is key to unlocking a new alternative to treating pathogens such as the Dengue virus.

               At the Biodesign Institute, researchers modify the N-glycosylation process that normally occurs in the Nicotiana benthamiana plants. Usually N-glycosylation requires a special lipid called dolichol phosphate to help attach a nitrogen molecule from asparagine with the glycan, a regular carbohydrate molecule found in plants. N-glycosylation is very unique because normally this enzymatic process occurs in the lumen, which is a network of membrane tubules and vesicles that are responsible for the production of hormones, of the endoplasmic reticulum in eukaryotes. This process is responsible for the folding of many eukaryotic glycoproteins and the cell-extracellular matrix attachment. However at the Biodesign Institute, N-glycosylation to enable proteins in the lab plants to produce the desired antibodies. Normally, since the plants are receiving genetic material from several other bacterial\virus species\strains the plant's immune molecules would attack the foreign cells\viruses. Due to our work at the Biodesign Institute, we were able to modify the glycoproteins' structure found on the surfaces of the introduced bacterial cells and viruses in order to mask their foreign identity from the plant and mammalian immune systems. Somehow by modifying N-glycosylation researchers are able to change the structure of the antibodies, that would be produced from the plants, so that these antibodies appear mammalian-like. As you can see these antibodies like the bacterial cells\virions will be able to float around a host's immune system and perform its duty without being detected by the host's immune system and being destroyed. Overall, the main job researchers want from N-glycosylation is for genetically modified antibodies to be synthesized that can be undetected from both the plants' and mammalian' immune systems.

                 That is the end to this blog but stay tune to news about the data collected from the antibody quantity produced from the Nicotiana benthamiana plants.   

Heat is the Answer to Cleaning

How to do-Autoclave?

                       Autoclave is a laboratory cleaning method where I have to place contaminated lab equipment into this oven-like machine to kill any bacteria, that would reside on the equipment. The lab equipment may include: beakers, flasks, graduated cylinders, lab bottles, and the lab bottles' caps. Before I put the equipment into the oven, I had to clean out all of the dishes using dish water and bleach so that I can kill as many bacteria as possible before the rest of the bacteria and their endotoxins are destroyed in the oven. Also, remember since I am working with glass beakers, flasks, and plastic bottles I will have to put aluminum foil to cover the ends of the equipment, so that the bottles, beakers, and flasks don't get damaged from the heat. Simply just like any household oven remember to wear oven mitts, because inside the autoclave machine is very hot. Don't be the person who gets burned from accidentally touching the inside of the machine. Believe me the machine is very hot and you can sustain a second degree burn from this machine! After I placed all of the lab equipment on plastic trays, I then place the trays into the machine and set the timer for about 2 hours. Lastly, once the two hours were up all I have to do is get rid of the aluminum foil and put all of the lab equipment away in their designated places. Pretty much that is all I did when performing this method. It is pretty simple enough, but remember to wear oven mitts, because I have already been burned from this machine and it sure hurt a lot!

Here is a picture of the inside of an Autoclave machine

How Important are Pellets in a Laboratory?

How Are Bacterial Pellets Made?

               On Friday, for the first time I learned how to grow bacterial pellets. The entire process was pretty repetitive and time-consuming, but overall the whole agenda was interesting. First off, I want to clarify what is a bacterial pellet and why we, researchers at the Biodesign Institute, need to grow them.

                A bacterial pellet is a clump of dense bacterial cells that grow together in a laboratory bottle due to a centripetal force. What produces the centripetal force, you may ask? Well, in the laboratory we use a centrifuge to produce this force as we spin down two or more balanced bacterial media bottles; in order to separate out the bacteria cells from the liquid media. The most important rule I learned about using a centrifuge is to make sure the lab bottles are balanced. By balanced I mean that both bottles have the same mass amount by using a common mass scale. In my case, whenever I needed to add more media solution into one of the bottles; the trick is to just add water into the less massed bottle, until the mass of the bottle is about the same as the other bottle. The reason why the bottles have to be balanced is so that the rotor in the centrifuge doesn't get damaged by spinning so fast where the unbalanced side of the cup holders doesn't teeter and totter, causing the rotor to break. In our case, we spun the bacteria at around 5000 rpm, which is very fast if any you get the chance to see it. It took around 10 to 15 minutes for us to spin the bottles down, but all the work was worth it since the bacterial pellet grew. See picture:

The brown patch in the picture is the bacterial pellet.

Notice how compact the pellet appears, where this tells me how the bacterial cells are clumping together due to centripetal force.

So far I have only talked about how the bacterial pellets grew, but now I still need to answer one of my questions about why we need to use them? When we grow bacteria particularly the Agrobacterium strain, we are focusing on bunching up all of the bacterial cells together so that we can have a huge concentration of these cells to infiltrate. Hence, the bacterial cells will clump together to form the pellet, as shown in the picture. The more concentrated bacterial cells we have then the more concentrated media solution we can produce to which the gene of interest, the gene that codes for the antibodies, can be encoded in the plants' tissue. In order to grow the media solution, I had to put the bacterial cells in a MES buffer solution, so that the bacteria can grow in the solution and be stimulated to transfer genetic material to each other. As the bacteria are growing in the solution, I have to then pour a little sample of the solution into a cuvette, which is a small container that fits into a spectrometer, so that I can place the cuvette into the spectrometer to measure out the optical density of the bacteria cells in the sample.  What the spectrometer does in order to give me the optical density of the sample is by passing a 600 nm wavelength light through the cuvette, where any light that was blocked by the cells themselves would be determined from the amount of light that was able to pass through the cuvette, which is measured out by the machine. The density value will then be used to calculate out an estimate of how much bacterial cells there could be in these large 500mL flasks, that contain the bacterial cell "waste" solution left out from the pellet.

Here is a picture of a lab bottle filled with concentrated bacterial cells after using the vortex

Whenever I use the centrifuge, the liquid that is still in the lab bottles where the pellet forms at the bottom is disposed into labeled bacterial strain flasks. The liquid may still has some bacterial cells in the solution, but most often then not it will be the MES buffer solution that I mentioned. I keep pouring in more solution into each specific lab bottle, until the pellet has formed completely. After that I can use a vortex spin machine to grind out the pellets to dilute the bacterial cells into a clear white liquid. That white liquid will be added to another solution of MES where then it can be used for infiltration. Other than that that is pretty much all the skills you will all need to know in order to make a bacterial pellet.

Here is a picture of a vortex machine.

              

How it's Like to Perform Agroinfiltration?

Agroinfiltration Firsthand

              In an earlier post, I have talked about what is agroinfiltration and how it is used in today's pharmaceutical companies to develop antibodies. After yesterday's lab day, I have finally performed agroinfiltration for the first time. Frankly, the entire process wasn't really difficult to perform but rather repetitive. All of the supplies I needed were a needle, a 10 mL syringe, several trays holding the plants, and a 100mL beaker filled with some medium solution. The medium solution looked clear and viscous, and luckily the solution wasn't harmful to the skin, because even with wearing laboratory gloves some media spilled on my skin. The quick solution to this occasion was to wash my skin with water, so like I said before the solution is safe. In the medium solution stores bundles of key nutrients that the plants need to grow and the Agrobacterium that hopefully will deliver the needed instructions for the plants to produce the desired antibodies. I used the syringe to pull out the solution from the beaker and with a needle I poked a small incision on the leaves' undersides. As you can see from the picture, next I placed my index finger on the top portion of the leaf and placed my syringe on the incision that I made from the needle, so that none of the medium flows out of the leaf. This process continues repeatedly until the leaf is completely filled with the medium, where as you notice this will end when the dark patch is spread all around the entire leaf.

Here is a picture of a scientist performing agroinfiltration by injecting medium inside the leaf.
Notice how the solution spreads throughout the leaf as shown by the dark patch.

              Like I said in my previous post agroinfiltration is an elaborate technique to  "convince" a plant's machinery to produce antibodies whose codons were delivered from another bacterial species. This technique is very simple yet can be missing when you are doing this for the very first time. Trust me I struggled a lot with making a small incision with the needle, because I would place too much pressure and end up poking a hole through the leaf. Other than that this method is very simple but can take a long time depending on how many plants you have to infiltrate. In my session, I had around 60 plants to infiltrate and that took about 2 hours for me to do with a lab partner, so this process is pretty long but repetitive. If anyone decides to perform this procedure, just remember to bring some headphones because this whole process can take a long time.

How VLP's Are Key to Future Vaccinations

VLP's Are the Key to Vaccinations

              Imagine a world where immunogenicity is offered to every individual all at a price of a vaccination shot. Thousands to millions of lives could be saved from deadly, epidemic diseases just by taking a single shot. Is it worth it? This is the world that I want to pursue and hopefully with the help of Nicotiana benthamiana  plants it will be possible. In this article that I have read, which is called Plant-derived Virus-like Particles as Vaccines, there are specialized particles called virus-like particles (VLP's) that are used to help the host immune system develop immunity against a certain surface protein or antigen. However, something to point out VLP's only help develop immunity in a host only against pathogenic viruses and not bacteria. These VLP's derived their unique structure from viral antigens that mimic the general structure of the real viruses, however these VLP's lack the viral genome. Due to a high presentation of viral antigens on a natural virus' surface, VLP's are able to copy these antigens and mimic the antigens on their surface. This result demonstrates that VLP's are valuable in vaccinations for humans, because our immune system just has to encounter these antigens for a first time, before our immune system develops antibodies that are best suited to those specific antigens' structure. The major problem to VLP's is the amount of production costs, but in this article a solution has been made.

              The lab plants, Nicotiana benthamiana, are a cheaper alternative to producing these VLP's, because of the low production costs, low maintenance costs, and high scalability factors that are offered by these plants. The production of VLP's in plants is a quicker process than other mechanism because the transgenic plants that I am working with grow quickly when placed in the right laboratory conditions: high humidity, water tank, fertilizer, and a healthy soil amount. It takes around two weeks for the plants to grow to the max controlled height that we want the plants to be up to, so that shows to tell you that these plants grow really quickly. What is so remarkable about these plants is that according to a theory about vaccine transportation; plants that hold the VLP's can be ingested in edible plant parts such as the leaves in order to transmit the VLP's particles orally to a host. However, this is only a theory and has been only tested in lab mice. Also, to point out there are several concerns over this delivery system such as possible denaturation occurring to the proteins that make up the antibodies due to the low pH in the human digestion system, poor recognition of the vaccine at mucosal immune effector sites, and antigenic tolerance. As you can see the biggest hurdle to using plants as an antibody production site is the delivery method of the vaccination to a host. In the article, the author mentioned that the most reasonable delivery method would be direct injection using a syringe needle, which is the most common alternative for any vaccination. In the next paragraph, I will be discussing how the VLP's develop immunity for hosts like humans against the actual deadly pathogens.

                  The way the VLP's are structured is the main reason why these simple particles can help hosts develop immunity against the wild type viruses. Since VLP's are particulate it allows them to induce T-cell mediated immune responses due to their interaction with the antigen presenting cells (APC) found in the host's immune system. Remember T cells are specialized immune cells that work to destroy a foreign cell by signaling the infected cells to undergo apoptosis, which means the cell self-destructs and dies. This action is vital to a host's defenses, because of how effective this process can be directed against pathogens. In order for the VLP's to induce the T cell activation they have to  mimic the process of a natural infection, where the antigens presented by the VLP's should trigger a cascade immune event where the host's immune system attacks the VLP's. As the immune system destroys the VLP's, since the VLP's have no mechanism to infect the host's cells, the B cells should develop "memory" against the specific antigen structures that were presented by the VLP's. In turn, the host's immune system would develop immunity against the actual pathogenic virus, since the immune cells can quickly develop an immune response against the pathogen quicker. Another reason that allows the VLP's to develop immunity in a host is the presence of epitopes on the cell surface. Epitopes are a specific region on an antigen that is recognized by the host's immune system and to which where an antibody binds to. The presence of thousands of epitopes on a single VLP aids to the processing and presentation of APC's.  What else is so interesting about these VLP's is that some of them are so small that they can diffuse to the lymph nodes, allowing the VLP's to be presented efficiently by B and T cells. That is all that is to it to VLP's and how they help develop immunity in hosts. These particles are very efficient at their job and hopefully become more available to be used in all future vaccinations.  

Why Does the "Position Effect" Matter?

What is the Position Effect?

Here is the pathway for transcription.

          There are two different definitions for the position effect that I would like to share in this blog. The first definition is that the position effect is a change in the expression of a gene after a chromosome's position is disrupted. Usually when a chromosome's position or a portion of it is then it's a result of translocation. A translocation occurs when there is a rearrangement of portions of a chromosome that take place between two nonhomologous chromosomes. Like any change in a chromosome, translocation can also occur due to a mutation, crossing over, or even mistakes in meiosis\mitosis depending on the type of cell (somatic or gamete). Not only that but the position effect can describe the variation of how a gene is expressed by identical transgenes. Notably the difference in the gene expression is due to enhancers that regulate other nearby genes. Enhancers are short DNA regions that are bound by proteins called transcription factors that stimulate transcription to occur.


                                                   Here is a picture that displays translocation
          
            What allows these enhancers to stimulate transcription to occur is because of its location near the promoter, which is the region of DNA that forms the site for which transcription can occur. What is so interesting about these enhancers is that they don't have to act upon the transcription site in order to stimulate transcription. Enhancers can instead be bound by activator proteins, which are specialized proteins that coordinate DNA polymerase and transcription factors to transcribe a gene sequence. However I almost forgot to mention that enhancers can be found in the introns, which are the noncoding gene sequences that become separated from a chromosome via RNA splicing, where because of this it doesn't matter if the enhancer is inserted into another chromosome (via translocation) because it will still affect transcription somehow. I haven't been able to find a reason why this still occurs. These enhancers are a really extraordinary topic to continue studying, because there are many enhancers that can be found in the human genome. One example is the enhancer called HACNS1, which researchers today believe had contributed to the evolution of the opposable thumb rotation and the ability to walk on two feet. These enhancers are the key topic that I believe everyone should continue to study, because there is a lot of researcher behind their possible functions in biotechnology.  

Here is a picture of the interaction between enhancers and the activator proteins.

The Importance of Agroinfiltration to Gene Delivery

What is Agroinfiltration?

                Agroinfiltration is a laboratory technique where scientists inject a suspension of Agrobacterium tumefaciens into a plant host in order to deliver a gene. The delivered gene would be expressed by the Nicotiana benthamiana host, where the plant host can synthesize the target protein(s). During this technique, Agrobacterium tumefaciens is injected into the plant host through either direct injection or vacuum infiltration. The reason why agroinfiltration is valuable to researchers is because many effective recombinant proteins can be produced over a relatively short time period. In the next paragraph, I will discuss the two different avenues to performing this technique.

              By direct injection, what I mean is that a scientist will have to grow the Agrobacterium strain in a liquid culture and suspend the bacteria into a buffer solution. After several hours, the bacteria should be placed in a syringe where the scientist can then inject the bacteria onto the underside of a leaf. Once the bacteria is injected the bacteria will enter the plant through the small openings of the stomata, that are located all over the leaf. When this whole process is finished, the bacteria should be reproducing inside of the plant, until there are enough daughter offspring for the bacteria to highjack the plant machinery to produce the encoded proteins that are held in the bacterial genetic material. Other than that that is pretty much the entire procedure to performing direct injection.

            Now in order to perform vacuum infiltration all you have to do is first place any leave matter, leaf disks, or even the plant itself into a beaker filled with the buffer solution. Wait until the leaves or whatever sample you are using is fully submerged in the solution and place the beaker into a vacuum chamber. Once the leaves are in the chamber turn on the vacuum, allowing the Agrobacterium solution to be forced into the stomata, which is a tiny opening in the plant tissue where gas exchange occurs, and into the mesophyll layer. This will allow the solution to be in contact with almost every plant cell that is found in that layer, which over time will give the cells the necessary genetic information to construct the encoded proteins.  

             How does the bacterial genetic material become processed? When the bacterium Agrobacterium tumefaciens gets injected into the leave, it remains around the intercellular space in order to transfer a combined gene with TI plasmid-derived T-DNA, which is the generic foreign DNA that the bacterium possesses. The bacterium will copy the gene as much as possible so that it can transfer the genes to as many plant cells as possible. The gene of interest will become transiently expressed, which means the genetic material is inserted into a eukaryotic cell in order to produce the target proteins, by all of the infected plant cells. After the genes become expressed, the plant organelles or "machinery" should be utilized to produce the target proteins. Once the proteins are made that is the moment during my laboratory where I can purify the proteins, which I discussed in an earlier blog, and harvest that protein, where in my case I want antibody proteins. Pretty much from there that is all that has to do with agroinfiltration. Believe me I  have discussed the different methods that are used in this entire process, so now I just want everyone to realize that all of the different methods I have discussed; all really come together in my project.  

            




  

How Today's Pharmaceutical Companies Expand Gene Delivery

How Lettuce Can Produce Recombinant Proteins (RP's)

         What is a recombinant protein you may ask? A recombinant protein is simply a protein that is made from the combination of several, different genetic material from other living\nonliving organisms. For instance, after reading Dr. Chen's article, Gene Delivery into Plant Cells for Recombinant Protein Production, I learned that the recombinant proteins he and his grad students are attempting to synthesize obtain their genetic material from mammalian, bacteria, plant, and insect cells. These proteins are responsible for the creation of the highly-valued antibodies that hopefully will help prevent viruses such as the Dengue and West Nile virus from replicating. As you can see, it is very important to study further about these proteins because they can benefit the medical world and hopefully bring an end to these disastrous epidemics. The process to producing a recombinant protein is rather simple, but really monotonous in the laboratory. Here is a diagram showing one way how recombinant proteins are made.

           Today's pharmaceutical companies have approved of the use of plants such as Nicotiana benthamiana to produce the recombinant proteins ever since the development of the first plant-derived therapeutic enzyme for Gaucher's disease. Gaucher's disease is a hereditary disease that causes a buildup of fatty tissue around the liver, spleen, and bones. The reason there are a buildup of fatty issue is due to a lack of production of the key fat break-down enzyme called glucocerebrosidase. The buildup of fat around the bones actually weaken the bone tissue and can cause an increase risk for bone fractures due to too much pressure on the bones. After this new treatment, enzyme replacement therapy, was discovered; the transition to plant gene delivery was seen more beneficial than ever. Gene delivery to the transgenic Nicotiana benthamiana plants is not only cheaper in terms of manufacturing costs, but the process is really simple. All I have to do is make sure the target transgene, which is the gene that I extract from another entity and transfer it to a target host to express it, is integrated into the plant genome. Like any other living organism it is at the genome where the genes become expressed and soon produce the proteins that are encoded from those genes' DNA sequences. However, recently a new strategy has been discovered regarding an even quicker way for the recombinant proteins to be produced from the transgenic plants. It turns out that recombinant proteins can be sequenced and produced by the transgenes directly, where the transgene being inserted inside the plant cell can manually use the plant machinery to produce the RP's directly. This process is done only if the 'position effect' is eliminated. I will talk about more about the concept of the 'position effect' in my next blog, because it is a very important concept to understand in my senior project.

           Lettuce is one example of a non-Nicotiana plant host that can produce recombinant proteins, however at a cost. Lettuce is known to produce higher levels of phenolics and alkaloids, which are compounds that can affect purification resins and hence add to production costs. Since there are a higher concentration of these compounds, there are a lot of FDA regulations that lettuce-produced recombinant proteins do not abide to; hence adding to more problems over manufacturing costs. For any con there has to be a pro. When lettuce undergoes the agricultural process called agroinfiltration, which I will discuss more about in my next blog, lettuce is able to produce efficient, abundant recombinant proteins. There is a study where researchers injected deconstructed viral vectors into normal, wild type lettuce hosts in order to observe the expressed pharmaceutical proteins. These researchers used a capsid protein that a combination of Norwalk virus (NVCP) and geminiviral vector genetic material to show that lettuce is efficient in producing recombinant proteins. The tested lettuce sample was able to produce the Norwalk virus like particle as efficiently as a Nicotiana plant host could do as well, where these functioning virus like particles (VLP's) can be used to induce an immune response in lab mice. As you can see they are producing a vaccine just by experimenting with commonplace lettuce that practically every individual can obtain from a farm.  The production of these VLP's are very beneficial to the production of vaccines, since their similar structural characteristics to the real pathogenic viruses can aid in developing immune responses against the pathogens in lab hosts and hopefully in the future for humans as well. Imagine a world where there is a vaccine for every single kind of pathogen. Lastly, since lettuce can be easily grown and organized in controlled acreage environments this adds to another pro to using lettuce as a VLP producer, and hopefully this manageable crop can be further studied to see if it can indeed create a VLP for every virus so that vaccines can be produced.   

           
  

Another Day in the Laboratory

The Amazing Application of Gel Electrophoresis

         Last Tuesday in the laboratory I helped made DNA gels using the technique, gel electrophoresis. In order to construct the gels, I needed two key substances, which were agarose and the buffer TAE. Without these key substances, there would be no way for me to be able to extract the DNA sample from a DNA plasmid. A key note I have to address about the plasmid is that we extracted the plasmid from the plant virus, Potato virus X. In this blog I will be talking about my experience performing the entire gel electrophoresis technique, and I will describe how the technique works.

         First off, gel electrophoresis is a laboratory technique used to separate out the DNA fragments of a certain plasmid based off of the DNA's size and charge. In my case, this technique is extremely important, because I have to separate out the junk DNA from the essential or specific DNA strands that is found in the plasmid sample. Usually the plasmid sample appears to be a clear liquid contained in a tiny test tube. Also to point out junk DNA are extra strands of DNA that an organism or nonliving entity such as a virus picks up from either the environment through the process called transformation or from other organisms or viruses through the process called conjugation. The specific DNA strands that I desire encodes for the construction of the antibodies that my whole project is entirely based on. As you can see this technique is very key to my project.   

  
Here is an example of a plasmid extract in a test tube

Here is a picture of some of the equipment needed to perform gel electrophoresis

               

           The entire process for gel electrophoresis is actually quite simple than most people think. In this experiment, I had to first prepare the gel by using agarose gels to show the DNA fragments according to the length of the individual fragments located on the wells. In my situation, I had to use around 1% of the agarose concentration to perform my gel electrophoresis experiment, because the wells that I had in the lab were really small while the DNA strands that I had were relatively larger than most DNA samples. I then insert the agarose gel into the casting tray and heated it using an electrical current, that was derived from a low voltage power source. Typically it took me around one hour to two hours, because of how low the voltage is in the source. It definelty took too long just for this one step but surely after I completed my gel the whole process I figured the entire process would be longer than I initially presumed. After the gel was heated, I grabbed a comb and placed it at the ends of the tray in order to make well holes on my gel.  After the gel is heated, I had to insert the gel into the gel tank, so that I can finally perform gel electrophoresis. I then inserted the blue TAE buffer fluid onto the gel until the surface of the entire gel is covered, so that the buffer can separate out any DNA and RNA molecules found within the sample plasmid. The key components of TAE that allows for it to perform this function are acetic acid, its Tris bases, and ethylene diamine tetra-acetic acid, where these different acids are responsible for sequestering out the positively charged cations. Remember DNA is typically negatively charged where by removing the cations evidently the DNA molecules are being separated from the RNA molecules. Once the buffer completely covers the gel I had to shift my focus to preparing the DNA from the plasmid sample.

Here is the comb "technique" I used.

Preparing the DNA

           This step is by far the easiest step to performing gel electrophoresis, because of how simple it was in the first place. Using a pipette, I inserted a known green dye into the DNA plasmid sample, so that the viscosity or tendency for a substance to resist movement of the DNA increases. This is very important for the entire process, because now the DNA, that will soon be inserted into the wells, cannot float out of the well and also somehow allows the movement of the DNA to be seen on the gel through fluorescent imaging. I then inserted a DNA marker pf known fragment lengths into the first well in order to approximate the size of the other DNA fragments in the other wells. The remaining DNA sample found in my plasmid test tube are then pipetted into the rest of the wells, and finally I am able to close the lid of the electrophoresis tank and play the waiting game. I have to say this part was very simple but really boring because of the long wait for the electrical current being transmitted from the electrodes, that are inserted into the tank.

Here is a picture of the electrodes being assembled onto an electrophoresis tank

How to Separate the DNA Fragments

              In gel electrophoresis, separating out the DNA fragments from the DNA sample is crucial to separating the different DNA strands such as the desired DNA strands that encode the antibodies and the junk DNA strands, that I don't need. Once I have the correct electrodes in their proper positions; I had to wait for the electrical current to be transmitted through each of the DNA wells, so that I can see if the DNA is migrating. Remember DNA is negatively charged so it would make sense for the DNA to be attracted to the positive cations that are surrounding the anode. It was really neat to see how the DNA is migrating from one well to another through monitoring the movement of the loading dye. Like I referenced before in order to monitor the movement of the loading dye; I can see the dye through fluorescence imaging. It was so neat to see the machine that performs this process, because truly it has done a lot of good for today's scientists than ever before. Overall this entire process took me about two days to complete, which basically shows how long it can take to do a simple yet intriguing process such as gel electrophoresis.

  Gel imaging machine

Understanding the Plant Anatomy is Crucial

Understanding the Plant Anatomy

          Today's lab work was very interesting, because I finally got to work with the Nicotiana benthamiana plants. Frankly, initially I presumed I was going to be able to study the plant production of the antibodies, but instead I was able to work on something more straightforward. I got to feed the plants and "groom" them. I do admit the term "groom" is the wrong diction, but frankly that is what I did today. First, before I started "grooming" and feeding the plants I learned more about the plant anatomy.  For instance, in today's lab work I learned that the test plants produce a lot of structures called suckers. These suckers are what give rise to the formation of a plant's stem. The reason why I had to remove these suckers is to control the height of the plants. Without the suckers the plants wouldn't be able to form a longer stem, hence the plants can't become taller. In my case, removing these suckers was really annoying because there were too many to remove on 60 plants! I swear even though these structures are miniscule, however I had to carefully remove them. Not only are they tightly connected to the plants, but I simply kept on removing the leaves with them. It was so annoying, but finally I got the job done with minimal damage to the plants.

Here is a picture of a Nicotiana benthamiana plant with its stem extending upwards

Here is an example of a sucker.

          After all of that trouble, I finally was able to feed the plants or simply pour a mixed water and fertilizer solution onto each plant. The process was very easy but yet messy at times. I don't remember exactly how many times I got splashed by the water, but I'm telling all of you my shirt was drenched by the time I finished. It was really extraordinary to see how much water we needed to water all of the plants. It took around eight to nine buckets filled with the mixed solution in order to water all of the plants! Those plants definitely receive more than enough water. Frankly, the whole process was really simple but too repetitive. I do have to admit plant biologists and researchers have a lot of work to do other than studying the plants themselves. Hopefully, more people come to realize that studying plants and their structures have more work than they think.  

How Plant-Made Biologics Will be the Future

A New Age in Antibody Production

               After reading the article, Plant-Made Biologics, it is very important to point out that Nicotiana benthamiana plants perform more functions other than synthesize the desired antibodies. These Plant-Made Biologic (PMB) plants are being used today to mass-produce several vaccines that can help combat infectious diseases being transmitted from the Dengue, West Nile, and HIV viruses. In the article, I found out that researchers use the domain III of the West Nile Virus protein envelope as a "vaccine candidate" for the West Nile virus. Somehow the N. benthamiana plants are able to synthesize antigen proteins that can be used to test the structure of the newly developed receptors, that the pathogens possess. If any of you don't realize that in order for a pathogen such as a virus to hook up on the surface protein, the antigen, of a target host cell; the pathogen has to latch its receptor onto the target's antigens. These newly developed antigens are responsible for the development of new vaccines, because whenever a patient is given a vaccine that individual is given a sample of foreign antigens that causes the individual's immune system to develop immunity by producing antibodies against those foreign antigens. Not only this but the plant-derived domain III somehow are able to resist denaturation, allowing the protein envelope's structure to not change, which allows the function of the envelope to remain intact. However a problem I have read from the article to this is that the vaccines produced from the domain III protein envelope haven't been able to work in humans just yet. So far the plant-made vaccines are being administered in mice.

              

  

What It's Like to Work in a Microbiology Laboratory?

            

              Ever Wonder What It's Like to be in a Laboratory?

As of Monday last week, I finally started my research in the laboratory. I have observed how the grad students at the ASU Biodesign Institute extract the necessary antibody proteins from the Nicotiana benthamiana plants and it is a very simple process. Simple laboratory techniques such as centrifugation, and filtration are used to breakdown the targeted protein from all of the biota, the potato X virus and the lab plants.

The first step I worked on in the laboratory was placing the fully grown plants in a blender in order to mash up all of the plant material into a droopy, green liquid paste substance. The reason why the grad students did this step was to acquire the necessary protein that has been stored in the plant material, which would be used to synthesize the antibodies. After all of the green "paste" is mashed up in the blender, it was neat to see that a simple everyday process called filtration is used. Simply the grad students just grabbed some filter sheets and placed the sheets onto four plastic, homogenous bottles. Filtering out the plant material was a very straightforward process in my opinion. The process is actually quite similar to filtering out coffee, when someone wants to have some tasty coffee in the morning. After all of the pure plant material is filtered and placed into four capped bottles, the grad students simply placed all four bottles into a plastic box and inserted ice to cover the sides of the bottles. It's pretty much the same when you place ice in a cooler in order to cool your refreshments. This step is the most essential one in the overall task, because like any protein there is an optimal temperature to which the protein needs to be in, so that the protein doesn't denature or break down in terms of structure. After 5-10 min the grad students grabbed the bottles out of the ice box and placed them in the centrifuge machine. The centrifuge machine we used was enormous. It looked almost like the size of a dryer! We set the four bottles inside the centrifuge machine and set the rotation rate of the machine to 1400 rev\min so that the protein found in the plant "paste" is layered and separated from the rest of the plant biomaterial. Thanks to all of these techniques I was able to observe how any plant protein can be separated from the plant residue, which allows me to further appreciate plants more.

My Goal in Epidemiology

What am I Doing?

        Hello my name is Armando Cancino. I am a senior from Basis Phoenix who wants to share to you what my senior project is all about. In my senior project, I am researching how bio engineered plants, Nicotiana benthamiana, can produce alternate antibodies that can be used to combat the dengue virus. These transgenic plants act like "test reservoirs," where I am able to genetically engineer these plants to produce different variants of antibodies. Normally, I don't know if everyone knows this but plants don't produce antibodies so by changing the test subject's genetic structure I will be able to convince the plants to produce antibodies.

         The reason why I am doing this project is to understand how researchers and epidemiologists  work in their every day career. I want to also receive experience working in a laboratory environment, because then I will be able to determine whether this field is the right one for me. Epidemiology is a very intriguing science, because like biology it is a combination of multiple sciences. There is no way for this science to be enclosed in one area of study, because this science works with other sciences such as biotechnology to fulfill its goals. For instance, in my project I want to understand how antibodies can be expressed from the plants machinery, which the genetic coding for the antibodies come from tobacco-mosaic virus, to prevent the dengue virus from replicating. I understand that one way antibodies prevent a pathogen from reproducing is through the process opsonization, which is the ability for multiple antibodies to bind to the receptors of the pathogen causing it to not be able to bind to other target cells. By this intuition I plan to find a way for these antibodies to be able to attach to dengue virions in mammalian cells, so that hopefully in the future people all over the world won't be harmed from the dengue virus. Hopefully my research will work out. Wish me luck!

This is the dengue virus

Why Use the Nicotiana benthamiana Plants?

Why Are Nicotiana benthamiana Plants Applicable?

After reading the article, The potential of plants as a system for the development and production of human biologics, I have found out that the reason why many researchers use Nicotiana benthamiana plants as a test subject is because of its many advantages. These plants provide low production costs for producing more test subjects and development of antibodies, high scalability in protein expression, low safety precautions, and like any plant there are very few plant pathogens that can be transmitted from the plant to a mammal. What else is more appealing about these plants is that at the Biodesign Institute there is no need for capital prohibitive facilities to store the plants, no need for any bioreactors, fermenters, or sterile delivery methods. Like any other plant these plants require carbon dioxide, water, light, and fertilizers to help them grow and develop. However a key point about these plants are that at the Biodesign Institute these Nicotiana benthamiana plants are considered transgenic, meaning that they are bio-engineered to express the production of antibodies by receiving viral genetic material, that codes for the antibodies structure, from tobacco-mosaic viruses such as Potato virus X. Normally, plants don't have any antibodies to protect their cells from pathogenic infections, as a result the plants that I am working with are synthesized to express the antibodies that can be used to fight off viruses such as dengue. The antibodies produced from the transgenic plants are tested in petri dishes where the researchers testing the antibodies can insert the dengue virus with mammalian cells into the dish in order to examine if the synthesized antibodies are able to disrupt the dengue virus from replicating. In the article, I have found out that the researchers insert mammalian glycosylation genes in order to synthesize antibodies that can coexist in both the plant's and the mammal's immune system. The researchers had to turn to this route, because the initial antibodies produced from the plants were destroyed by the mammalian immune cells, that were found in a separate petri dish. Through the process called N-glycosylation, which is basically attaching an extra sugar molecule called a glycan to a nitrogen atom found in the amino acids of the proteins in the plant cells that produce the antibodies. Through this process the results were successful as the plant-produced antibodies were able to be transported into the mammalian cells to help defend the mammalian cells from the dengue virus.     

Here is a picture of potatoes having Potato Virus X



Meet Potato Virus X

Intro to the Dengue Virus and the Nicotiana benthamiana

What is the Dengue Virus?

The dengue virus is a RNA positive flavivirus that is transmitted to humans through mosquito bites. By the definition of a flavivirus it has a protein envelope and a coccus (circular) shape while also being 40 to 60 nm in diameter. This virus' genome is composed of single-stranded positive RNA, allowing this type of virus to produce its proteins directly from the template RNA strand. The template strand for any organism's genome is the coding sector for the production of proteins in any living or nonliving (viruses) organisms. According to the CDC around 400 million people have been infected by this virus particularly affecting people at the tropical regions. The dengue virus is known to cause a severe fever, headaches, joint pain, low white blood cell counts, and bleeding manifestations around the gums, nose, bruising, and vomiting of blood. The dengue hemorrhagic fever (DHF) is the key symptom that is associated with the dengue virus, where the fever lasts between two to seven days. The fever can cause blood vessels around the infected area(s) to become permeable or "leaky," which causes blood to escape these vessels and leak into the peritoneum, the membrane that surrounds\covers the abdominal cavity and the abdominal organs. Once the blood enters the peritoneum an accumulation of fluid (in this case blood) can envelop in the abdominal cavity, causing swelling of the abdominal region. Over time as the abdominal region becomes swollen up somehow the circulatory system can shut down, causing the infected organism to not be able to transport oxygen around its entire body, which ultimately leads to the organism to its death.
The dengue virus epidemic has been occurring since the 1950's, due to increased mosquito populations at the tropics. I have found that the reason why there are increased mosquito populations around the tropics is because during the 1950's there has been increased rainfall precipitation around the tropics due to rising sea level. I believe that the rising sea level is caused by global warming because throughout the 20th century the Earth's temperature has been increasing, due to the destruction of the ozone layer. The ozone layer is the section of the atmosphere that acts as a sheath, surrounding the Earth and reflects UV radiation from the sun from impacting the Earth. However due to the 20th century technological changes; the ozone layer has been getting destroyed due to the accumulation of green house gases such as methane, DDT, nitrous oxide, carbon dioxide, and etc. Greenhouse gases such as carbon dioxide and methane are accumulating in the world due to increase in industrialization such as the production and operation of cars, factories, generators, and so on. As the ozone layer is depleted, more radiation can enter the Earth causing the Earth to become warmer. Glaciers found in the Artic and at Antarctica are then melted from this increased heat wave, which ultimately causes the sea levels to increase.
In my project, I will be testing Nicotiana benthamiana plants to see if these plants are able to express effective antibodies that can be used to stop the dengue virus. Nicotiana benthamiana plants are common herbs that can be found at Australia and grow from 0.65-5 ft. tall. These plants are very useful in my project, because they are easy to maintain and they have minimal contamination pathways. In my project, I plan to use these plants as a "tool" for me to develop antibodies in my genetically engineered plants. I want to produce antibodies that are suitable to prevent the dengue virus from infecting mammalian cells, so that the dengue virus isn't able to reproduce. If I am able to produce these suitable antibodies, then I can test them on a petri dish filled with grown, incubated mammalian cells where I inject a dengue virus strain into the dish. If the antibodies are working, then what I should observe is a low dengue virus count. Imagine if there are antibodies that can stop every dengue virus strain. Scientists would be able to inject these modified antibodies into mammalian organisms such as humans to fight off the dengue virus. Who knows how much lives can be saved from this discovery?