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.
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.
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