The Complicated Matter of Ti Plasmid

It took me several hours to seek out relevant information and piece things together in a coherent fashion because online information can be rather vague at times or inappropriate so read with patience.

Background

Let's look at the Ti plasmid in its natural form:

As mentioned in class, it has two main regions - the T-DNA (transfer DNA) and non T-DNA region.

T-DNA

T-DNA consists of 3 parts you should at least take note of:

1) left and right borders (about 25 bp)

These would be where the products of the vir genes (see later) will nick to splice out the T-DNA

2) opine synthesis genes

Opines when expressed in the transformed plant cell are nutrients for the bacteria Ag. tumefaciens

3) tumor producing genes

In this category you will find many reference to genes that encodes enzymes which control the synthesis of auxins and cytokinins. Some sites will simply refer to the genes for auxin or cytokinin production.

(3L: I was right after all! For a moment I was torn between their true nature: do the genes encode for enzymes or for the protein)
Non T-DNA region

Here is where you find the OriR and Vir genes. Vir or virulence genes serve to aid the transfer of T-DNA to the plant cell.

Alright if you insist.... on the diagram above, you also see opine catabolism gene yah? Because the opines produced will be metabolised by the bacteria for enerfy and are of no use to the plant.

--------------------------------------------------------------------

Recombinant form of Ti Plasmid.

In this diagram, we can see that the tumor-causing genes and opines synthesizing genes are removed and replaced by your gene of interest e.g. gene for Bt toxin and plant selectable marker e.g. kanamycin resistence gene.


Note to point: The plant selectable marker and gene of interest are coupled!

(This is what I have to confirm because there are too many possible combinations/methodologies in the market right so the the simplest one (I think) is to treat the two as coupled to each other. In other words, if a transformed cell exhibited kanamycin resistance, we can safety say that the gene of interest is also present in the cell. Okie?
This would resolve the issue Slagoon has on having a plant cell that may exhibit kanamycin resistance but does not have gene of interest.)

You will realise the presence of a BACTERIAL selectable marker e.g. antibiotic resistance as well, outside the T-DNA region. That one is like your selectable first marker we used to talk about. When the plasmid is induced to enter the bacteria, this marker will allow one to know if the bacteria has been transformed.

At this point in time you might ask(or I rather) : can we use any antibiotic resistance marker in both cases?!
NO!! Try not to at least.
Kanamycin (or neomycin) resistance gene is highly popular as a Plant selectable marker because it is rather toxic to the plant cell, coupled with the fact that it is not used in medical treatment (thus lower the risk of cultivating kanamycin resistant bacteria in our gut when we eat the transgenic plant, and the fact that the gene might subsequently spread through horizontal transfer).

For bacterial selectable marker, we can still go back to ampicililn and tetracycline (these are not usually used for plant because they are less toxic to plant and also the fact that they are very common in medical treatment)

-------------------------------------------------------

And so the plasmid get transferred.
Note that only the T-DNA get transferred and not the entire plasmid. The T-DNA is spliced out and chaperoned to the plant nucleus by enzymes and proteins (respectively) which are encoded by the vir genes.

Then TaDA! you get a transformed plant cell that will survive on an agar plate full of kanamycin and has the gene of interest!

I will leave the growth of this plant cell to you. ..



Nites! It is Friday night!Zzz.........