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Full Version: Genetically engineered papaya - agrobacterium or gene gun?
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I am writing a paper on the genetically modified papaya (55-1), engineered to contain a coat protein gene of the papaya ringspot virus.. As far as I understand, this gene is used for cross protection (use of a mild virus variety to protect the plant against damage caused by the more severe variety of the same virus)

Here is my question! I have to explain the process of how it was engineered, and so far found information that describes how a agrobacterium tumefaciens plasmid was used, containing the coat protein and a few antibiotic selectable markers. However, in the same paragraph, I also read that the plasmid DNA was added to the papaya genome by "particle bombardment" - which would be the use of a gene gun.

So, is it possible to use both methods or am I misunderstanding the process?

Also, could someone explain to me what exactly promotors and cassettes are and how they are used to engineer the plant?

Thank you so much!

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First of all, particle bombardment does not necessarily mean that the gene gun is used. Gene gun itself represents only one way to transform the cells of interest using biolistic method. It can also be done using special type of machine. These machines do the job by using high-pressure helium pulse and the target DNA which is coated onto high-density microparticles (usually gold or tungsten). The whole process is done in partial vacuum which reduces the friction of the particles as they move. While the gene gun is more practical, the machine performs better job and it is able to work under more diverse conditions (for example, it tolerates wider range of humidity).

As for the usage of both methods simultaneously (agrobacterium and particle bombardment), it could probably done but on different specimens because we know that any kind of transformation is not 100% efficient (meaning not all cells will be transformed), so maybe two methods can be used to get better results.

Now, the promoter is the region which is responsible for the initiation of transcription (RNA polymerase binds here), so it must be present near our gene in order for it to be expressed in the host cell. Different kinds of promoters can be used and the choice depends on the goal, because the level of expression can be affected with specific promoters. So if want to engineer the cells to be able to produce certain protein in high amount (like insulin, e.g.), we will probably use high-expression promoter. On the other hand, if our target gene codes for the product that might be toxic to the cell in high amounts, we will use low-expression promoter.

As for the cassettes, they are kind of independent DNA sequences which contain certain genes. They are mobile, meaning that they can move from one place to another in the genome, but they can also exist as a separate circular DNA molecule. The thing is that they do not contain promoters, instead, they integrate themselves into the genome in the places called integrons (which are basically the docking stations for cassettes). Once the cassette has “settled”, it can be replicated from a nearby promoter.