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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.


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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Welcome! This is the Supporting Content section for the Target Nucleus Page | Carbon Free Footprint Project of the MMINAIL.

The Concept Library of the MMINAIL is a static informational website.

As such, a Supporting Content section is placed below each (.html) page in the flow of the default layout to grant the end-user additional pertinent information when navigating this site.

E Books

With today's explosion of modern devices, some consumers like to read their articles on their Kindles or iPads.

Therefore, a method to convert any page of this website into an eBook is auto-built into the top of the Navigation aside as well as in the footer of every page.

Simply click the following eBook icon = small dot epub button eBook icon to render the current page as an Overdrive (.epub) or a FB Reader (.mobi) .

Note. The Apple iBooks platform also reads and displays the (.epub) format, as well.

Secure Hyperlinks

The A's Have It!

In today's complicated world of coding, even the use of the standard hyperlink ...

    <code><a href="" title="" target=""></a></code>

Is placed into question by the standards of newer, more modern browsers such as Google's V8 powered Chrome browser.

Safe Internal Links

Therefore, to make it easy for the end-user ...

The MMINAIL will show safe, internal links in the bootstrap default color of light dodger blue.

Safe External Links

In addition, safe external links when designated https secure ie.) https:// ...

Will also be shown in the color of light dodger blue.

Standard External Links

All other external links designated with standard http ie.) http:// ...

Will be shown in the color of indian red.


For an acronym to be visually effective when reading a line of text, the acronym must first be declared.

In addition, the acronym must stand out from the body of information.

To accomplish both of these objectives, the MMINAIL has selected Ashley Gold.

Ashley gold is a primary color within the registered Logo Badge of the Benefit corporation.

Whenever the end user spies a designated Acronym of Ashley Gold ...

Simply hover over the Acronym to reveal the underlying meaning of the acronym embedded in the title element of the abbr tag.


The abbr tag can also be used to house Definitions as well as Acronyms as we have seen in the above referenced Ashley Gold.

Whenever the end-user spies a designated Acronym or Definition of Ashley Gold color ...

Simply hover over the Acronym or Definition to reveal the underlying meaning embedded in the title element of the abbr tag.

How To Navigate This Website

At this website, we have the Html Hub for the Benefit Corp aka our Concepts Library.

You (Usted), as a current visitor of our Concept Library, are now at the Target Nucleus Page | Carbon Free Footprint Project page.

Navigation Aside

In the Navigation <aside> section of this website ... ➡️

The end-user will find a Stack of Topics.

Stack O Topics

  • The Navigation <aside> to your right houses the Stack of Topics ... ➡️ .

  • At each Topic, the end-user will find a hyperlink back to the Home page of the Concept Library.

  • By clicking on the Home icon   the end-user will be returned to the Home page of the Concept Library.

  • To select any of the other Topics or subtopics, simply click on the corresponding glyphicon to open up the referenced file.

  • If you get stuck, simply click on the Home icon   to be hyperlinked back to the Home page of the Concept Library.


When an end-user clicks on a Topic of Interest over at the Stack of Topics in the Navigation <aside> ... ➡️

The menu explodes vertically to reveal several Subtopic choices.

Learn Topic

A Learn Topic Subtopic selection exists under each Topic of Interest.

The Learn Topic Subtopic selection sits just above the Home icon   Subtopic selection divider for each Topic of Interest.

Note. You may click on the Home icon  Subtopic selection at anytime to be hyperlinked back to the Home page of the Concept Library.

Got An Idea?

If you have an idea that will spruce this site up even better than it already is, then you know the drill ...

Simply go ahead and fork this repo, make your changes, and send us a pull request.

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25 Mile Rule

The MMINAIL is dedicated to the preservation of the hard-earned rights of Medical patients to grow up to (12) state-legal Medicinal plants.

In a connective cartography of ideas and hyperlinks spanning the entire connectome of the modern Medical Marijuana Initiative ...

The open source authors of this project hope to enlighten the world to the plight of the common medical patient.

Due to a labyrinth of government regulations and costs, today's common medical patient is virtually devoid of healing phyto-cannabinoids and other beneficial terpenes globally for no other reason than his or her current residential address.


"When residing 25 miles extant of a state legalized dispensary"

Doesn't that sound a bit strange to you?

Restricting access to medicine ...

Almost Un-constitutional, isn't it?

And, with patients in Arizona paying anywhere from $75 to $90 USD for a (1/8) ounce of medicine ...

Don't you think it is time that we allow ALL patients the right to grow, stash, and cure their own medicines?

Make Contact

We do at the MMINAIL.

If you agree, why don't you donate a little spare cash?

Or, a little of your coding time?

Or, simply contact your favorite Arizona State Senators and Congressional Representatives today!

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However, the information contained in this website, and in its pages, is offered to the public without any warranty expressed or implied.

Therefore, the author of this website, and by extension its pages and content cannot be held liable for any damages that may be caused indirectly or directly by the software instructions or tutorials contained in the pages of this website.

For further review, please see the License page of this website and/or the Privacy Policy page of this website to clarify.

Reproduction In Part

Most authors will allow the reproduction of their works in part when the case of brief quotations embodied in critical articles or reviews is addressed.

Therefore, please remember to provide a Link-back to the original author, or to the publisher of the original publication when citing.

Thank you for your consideration.

Coding Community

If you are an expert in your field of specialty ... Why not fork the working repo of this Website and create a Sub-menu of your choosing?

Send us a 'Pull Request'. Our team of coders will review your contribution and get back to you with a "thumbs up" ( or, "thumbs down" ).

Can You Contribute Code?

As we can now decipher the time spent by our functions at the code block level ...

How much of your coding time are you willing to invest in this project?

As we rely heavily upon contributions from the coding community, your contribution of code will be greatly appreciated.

Can You Contribute Cash?

As we can now spend the equivalent of our grandparents' monthly mortgage payment at our local Sunday football games ...

How much of your spare cash are you willing to invest in this project?

As we rely heavily upon contributions from the coding community, your contribution of cash will be greatly appreciated.

Can You Contribute Ether?

As we have witnessed the exponential growth of Bitcoin (BTC) from 9 cents USD per coin ...

To over $18,000 USD per coin over the past nine (9) years.

Plus, the recent explosion of Ripple (XRP) from less than one-penny to over $2 USD.

How much of your spare Ether ( ETH ) are you willing to invest in this project?

As we rely heavily upon contributions from the coding community, your contribution of ether will be greatly appreciated.

Coinbase Public Hash

Scan the following QR Code to extract the Coinbase Public "hash" address in support of our Medcoin™ Crypto Currency Project.

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Carriage Return

The carriage return, or ➡️ Is used extensively throughout the working repos of the Concept Library.

Screen Responsiveness

This page has been tested for mobile responsiveness on a virtual Galaxy S5 smartphone emulator.

The authors have determined the accuracies of the page renderings at both a portrait width of 360px and a landscape width of 640px.

In addition, pages are also tested against an Amazon Kindle Fire HDX 7, 3rd Generation tablet.

On the Kindle tablet, both portrait and landscape renderings appear accurate, as well.

And, on the Amazon Fire phone, using automatic screen rotation, both portrait and landscape renderings appear accurate.

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