### Target Nucleus: Using A. tumefaciens as a Vector for Introducing the Over-expression of Limonene in Cannabis.

Welcome! This is the Target Nucleus page of the Carbon Free Footprint project.

The total area of cultivated land in the year #2004 was 1,400 million hectares ( 1.4 billion hectares ).

Surely the world's scientists will find a way to muscle in a little Cannabis plantation here or there throughout the globe in our current millennia.

More to come ...

#### A. tumefaciens

A. tumefaciens is a common bacteria that causes crown gall disease.

Dicots are especially susceptible, both plants and trees, though Monocots may be infected, as well.

Dicots are 'di-cotyledonous' woody shrubs and trees that have spread across the globe.

Dicots have broad leaves with branching veins like an Apricot tree.

The serrated leaves and branching veins of a Cannabis plant, though an annual herb like the Basil plant, also qualify for inclusion in the family of Dicots.

On the other hand, narrow leaved plants with parallel grains such as grasses, grains and cedar trees are known as Monocots.

Monocots are 'mono-cotyledonous' plants that have spread across the globe, as well.

#### The Magic of Bacteria

A. tumefaciens does its magic by way of a 'Tumor Inducing', or TI plasmid.

A plasmid is a circle of DNA that contains genes, yet is separate from the parental chromosomes of the bacteria and can, therefore, be replicated on its own.

The size of the TI plasmid is between 200 and 800 kb ( base pairs ).

However, to perform the infection, a region of between 12 and 24 kb ( base pairs ) is used.

This 'Transfer DNA' region, or T-DNA is integrated directly into the nucleus of the host plant during the infection process.

The goal of the integration is to change the parental chromosomes of the host that are present in every nucleus, of every cell of the host organism.

But, here, specifically, the cells of the growing organism in the crown area between the trunk and the root system are targeted by the bacteria.

One function of the genes that are being integrated into the host genome is to form the visible gall structure.

A second function of the genes that are being integrated into the host genome is to synthesize opines.

Opines are modified Amino Acids that manufacture both carbon and nitrogen molecules that can now feed the expanding colony of A. tumefaciens.

The host plant now spends its ATP energy packs creating a food for the bacteria, in a form of which the host plant itself cannot use.

This is a naturally occurring state of hijacking of the bio-chemical machinery of the host plant by the bacteria!

Outside the transfer region, on the polar opposite side of the plasmid ring, are genes that act upon the process of opine catabolism.

This assisting 'Virulence Region' of DNA and its compliment of genes sits on the plasmid ring, as well, but far away from the T-DNA transfer region.

Note. Opine catabolism can be enhanced through the manufacture of enzymes that break down the opine.

Pretty clever, heh? The bacteria retains the genes for manufacture of the enzymes responsible for breaking down the opine away from the integrating transfer region of the plasmid.

Therefore, the host plant is not able to break down the copious amounts of opine that the tree is now genetically forced into making!

Also outside the transfer region of the plasmid, are the genes responsible for encoding Virulent proteins ( Vir-p ) that facilitate the transfer of the T-DNA snippet through the transport channel.

Thus, the integration apparatus assisted by the the Vir-p is able to transfer the TI region of the plasmid into the host plant via the transport channel outside the purview and control of the host plant, as well!

Gotcha! Punch that plant cell wall and membrane!

Target: Nucleus!

#### Hitching a Ride

Two can play this game of hijacking the genome!

Why not isolate the gene responsible for kicking in the manufacture of Limonene, an un-foreign substance naturally resident in the Cannabis plant, to the point of Over-expression?

While inserting the gene for the Over-expression of Limonene, why not add a marker that will eliminate all copies of the target genome not successfully integrated?

This can be achieved by way of another, simultaneous gene insertion.

Specifically, the gene that induces resistance to the anti-biotic ( bacteria killer ) Kanamycin.

Kanamycin is also toxic to plant cells.

By incorporating Kanamycin-resistance into the genome of the host plant, those plant cells not affected by the transfer plasmid will wither.

Upon application of a dose of Kanamycin, introduced genes that manufacture enzymes that can in effect 'disarm' the killer Kanamycin will move into play.

Thus saving your preferred callus from certain death!

Any explant not containing the genes for Kanamycin-resistance, and by extension the genes for Over-expressing Limonene, will be destroyed by the application of Kanamycin.

Pretty final, heh?

Think of that the next time your daughter decides to date a plant biologist.

Cold!

#### The Knockout

Why not 'knockout' the tumor producing genes, the virulence region, and the genes coding for both opine synthesis and opine catabolism from the plasmid prior to integration with the host plant?

Good idea!

Let's do just that!

By 'knocking out' the tumor producing genes, the virulence region, and the genes coding for both opine synthesis and opine catabolism we have created a benign plasmid.

One that can be loaded with the genes to Over-express Limonene, in addition to the identifying Kanamycin-resistance genes.

Simply place the genes into the transfer region ( T-DNA) as described.

#### Don't forget the Vir-p's!

Remember, the Virulent proteins, or Vir-p's assist the transfer of the entire plasmid by facilitating movement of the plasmid through the transfer channel between bacteria and host.

Toss a few of those into the balance of the plasmid for good measure.

By conjugating a common E.coli bacteria containing the target plasmid with a knocked out A.tumefaciens bacteria containing only a second 'Helper Vector' ...

One that is full of the genes required to replicate Vir-p's, the now combined A.tumefaciens is ready for injection into the host plant.

Note. The genes required to produce the Vir-p's will be activated upon insertion.

The virtual goal of the balance of this report will be to reproduce a single Transgenic Cannabis Plant, or TCP phenotype capable of producing Limonene.

Next, a True-breeding line of Cannabis plants that truly Over-express Limonene via the introduction of a promoter sequence into the genotype of the line will commence.

Finally, a synthetic version of the Over-expression gene containing DNA triplets more common to plants will be introduced to the line resulting in a 100-to-1,000 times increase in the manufactured expression of Limonene by the plant.

The true-breeding line of TCPs can then be propagated from the lab via Explant Clones, or seeded from fresh achenes after further back and cross-breeding.

The extraction of Limonene and other terpenes and cannabinoids from the herbaceous plant material of the Cannabis plant by mechanical methods utilizing CO2 under pressure are now a reality in today's modern laboratories.

Scaling up our processes to accommodate a growing market for pure Limonene and other novel terpenes and cannabinoids is the wave of the future.

More to come ...

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