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