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.