Author Topic: Genetic Modification at home  (Read 454 times)

Sedit

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Genetic Modification at home
« on: May 13, 2010, 02:58:35 PM »
Disclaimer: This thread is very half assed and I have been working on it for a while but due to an extreme lack of focus I have decided to just release it as is and let it grow from there. If I have misplaced or mislabled references which im sure I have im sorry. There is much more material I have to add to this thread but if I don't put it out there now I may never do so. I chose this option because there appears to be a HUGE amount of voodoo associated with Genetic modification when viewed by those who are outside of the circle so to speak. I have done relatively little research into it and my views on the difficulty of GM have changed dramaticly.


The idea of Geneticly modifing plants always seemed like a pipe dream to me until a short time ago when I realized the potential of a new form of modification that can be performed using a common soil bacteria and the roots of various plants to produce large quantaties of secondary metabolites from common starting materials. This thread over at Blacklight peaked my intrest in the subject sometime back and there are a couple good references to check out pointing at the possibilitys of Genetic modification on an amature level. I am by no means an expert in this field but I will try my best to explain in in a fashion that people can understand to the best of my understanding.

Hairy Root Cultures(HRCs) are the result of an infection of a typical root by the invasion of an Ri Plasmid containing bacteria. These bacteria contain a circular form of DNA known as plasmids with the ability to splice itself into the normal structure of plant cells leading to something that resembles a tumor in structure and growth in many instances. Once the correct form of infectant is identified and isolated it is use to infect the host root in an attempt to produce a culture of hairy roots. These can then be cultured in a seperate Bioreactor simular to aireated brewing equipment shown by the text SECONDARY METABOLISMOFHAIRYROOTCULTURES IN BIOREACTORS[1] to produce a large amount of high cell density rootmass in a relativly small area. Once cultured the reactors have been known to keep the DNA stable for over a period of 4 years as evident in Link showing how a root was made to glow by inducing the DNA of a jellyfish to fuse thru the HRC process for over 15 years in some cases(Floresetal.,1999)

What is needed is to isolate a viable strain of bacteria that contains the correct root inducing(RI) plasmid which is primarly contained in the Agrobacterium rhizogenes soil bacteria. If you look at Complementation of Agrobacterium tumefaciens[2] you will see the obvious infection site in the root is disinquished by the large nodual on the root. If anyone has seen roots in the wild they will know that this sort of infection appears fairly common and first order of business would be isolation of the correct Agrobacteria to induce the formation of these Hairy root cultures. The lumps on wild infected plants seems like the best place to take a swab from to start isolation of the bacteria.

Lets jump ahead and assume we have isolated and cultured the root we wanted. Its at this point that a simple areated nutrient solution simular to how hydroponics works with the addition of some bioprecursor to the mix and these will convert large amounts of starting material to a finish product increasing concentrations well beyond the normal for a plant. These HRCs do not seem to suffer from the same metabolyte toxicity that plants do and as long as the precursor is feed in the reactor it will continue to produce the secondary metabolytes without death to the plant as is normal in nature. The most obvious use of this is the conversion of Eugenol to Safrole quickly and cleanly


You can see the simplicity of my micro "Bioreactor" as nothing more then an aireated tube filled with water and at the moment a Rather crude but if I didn't start somewhere I would never start. My main goal is to keep this little clump of grass alive for a long enough time without infection to prove its ability to run for extended period of time. This at the moment is nothing more then a form of hydroponic growing equipment and I think all I will feed it is a few ML of Suger solution every couple days since I don't think for these test it needs much more. In all honesty im trying to feed any bacteria present more then im trying to feed the grass because I want to see what kind of hold Fungi or bacteria might take in such an experiment.  The initial weight of the grass is 2.3 grams and I will measure it after im done as well to see if it grows any at all thruout the course of the experiment. I do not have much faith in the experiment at the time but my main goal is to proceed to the stabalization of a  culture of Rootbark from a sassafrass trees. I don't think a Hairy root culture is fully needed to test what I had in mind since the reaction need only a single enzyme in the conversion of Eugenol and not a multistep process that many chemicals needs involving the entire root.The lump of grass seen in the picture was kept alive under water for over 2 weeks and it was not until I stupidly refilled it with tap water when the solution started to evaporate did the grass die. Keeping the roots alive after getting it started I think would be fairly simple in a home setup.


Lets for a second take a step in another direction. HRCs are just one form of tissue culture and with simple techniques explain in reference [] you can produce things like Codeine, morphine ect...
Quote
Production of fine chemicals like Codeine, Diosgenin, Sitostero, Quinine, Vincristine, Atropine, Pyrethrin, Saffoon and Methanol which are presently produced from plant sources can be produced from cell culture. However, the commercial success has been obtained in production of `Shikonin' from cells of plant lithospermusm erythorhizon.
Image in your head the potential of a tissue culture from the Morphine producing areas of the Poppy plant. A steady stream of waste liquor containing the substances desired could be saved and concentrated on an unlimited scale. This is all not to mention the potential that a tissue culture from the budding female pot plant could have.

Odds are this does also have the potential to make a large amount of cacti from a very small second of the source cacti.


This is where I began to lose focus and intend to finish reporting the subject and slowly cleaning up this thread as time permits. If you noticed missing links or misplaced references tell me and I will try to find them since I have more but am insure if I have linked to all of hem.



Further reading:
http://openpdf.com/ebook/hairy-root-pdf.html

Recieved references:

  • [1]SECONDARY METABOLISM OF HAIRY ROOT CULTURES IN BIOREACTORS

  • [2] Complementation of Agrobacterium tumefaciens tumor-inducing aux

mutants by genes from the TR-region of the Ri plasmid of
Agrobacterium rhizogenes.


External references
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Naf1

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Genetic Modification at home
« Reply #1 on: May 14, 2010, 12:34:56 AM »
So that was the good news! Vesp had pulled me up on it earlier, which is why I have all this on hand!

"These bacteria contain a circular form of DNA known as plasmids with the ability to splice itself into the normal structure of plant cells leading to something that resembles a tumor in structure and growth in many instances."

Vertical Gene Transfer
When you are born you receive your own DNA (genes) from your ancestors (parents ect, dictating your height, eye color, hair color, and everything else) this is called vertical gene transfer. And after Gregor Mendel, discovered how that works, there has been many since to take advantage of that we had plant breeders like Luther Burbank (and DJ Short lol) have used those rules to produce our exceptional crops(did you know Luther Burbank created over 800 different strains of fruits, flowers grains grasses and vegetables using the laws of Mendelian inheritance the most interesting is how he bred a spineless cactii for feeding cattle that were starving at the time. He was able to realise that the spines had evolved over time as protection from grazing animals, so his breeding program was aimed at reversing the genetics instead of advancing them in this case!). The obvious limitations of these methods are you need to sow large fields of the plant in question to select the best genetics to start with, then cross with the selected pollen. Then grow out a massive field of the seeds produced in the controlled pollenation, these plants are then rigorously screened and only the best hundred or so kept the other (20,000 discarded) these are pollenated again with carefully selected pollen and process repeated until about F5 or F6 a plant that will reliably produce the qualities you selected for is backcrossed against one of the originals to produce F1 hybrids with the new quality. This takes alot of space money and especially time!

Chemical Mutagenesis
Then came mutagenesis, which was discovered in the 1920's that x-rays caused mutations in fruit flies, and also the same effect on humans. The interaction of certain environmental chemical compounds and cell metabolism may result in genetic changes in DNA structure, affecting one or more genes. These chemical-induced mutations are known as chemical mutagenesis. Most cancers and other degenerative diseases result from acquired genetic mutations due to environmental exposure, and not as an outcome of inherited traits. Chemicals capable of inducing genetic mutation (i.e., chemical mutagenes or genotoxic compounds) are present in both nature and man-made environment and products.

That turns out to be a massive short cut to traditional breeding techniques, as you can alter the DNA there and then, instead of waiting for a natural mistake in the DNA to produce mutants or breeding out massive amounts of plants to try and find the odd plant with desirable traits. You can treat a plant and be sure the offspring that survives all will be altered in some way (some bad some good). Thereby planting 10,000 normal seeds you would produce maybe 10 plants if you were real lucky with new desirable traits, 10 with the opposite(undesirable) the majority (9,500++)would be uniform (same as parents) and the others with changes so minor not worth worrying about. Whereas you plant out 10,000 mutants and 10,000 will be completely different in different ways from the parent! Ranging from growth differences to alkaloid biosynthesis.
And those techniques are still used today! It is more of a pot luck approach than the recombinant DNA techniques of today, but the basic methodology is the same; Treat a large field of plants with a mutagen, which randomly changes the DNA of the plants that survive. Then pollenate with carefully selected not mutant pollen, plant out all the seeds produced with changed DNA and monitor the mutations. Testing to see if there are any usefull novel compounds produced, if alkaloid levels are super high (or just as likely super low). Alot would not even grow properly ect.

PRODUCTION OF THEBAINE AND ORIPAVINE
JOHNSON & JOHNSON 2002
A method to improve the thebaine and/or oripavine yield of a stably reproducing Papaver somniferum, the method comprising the steps of: a) exposing at least one poppy seed of Papaver somniferum to a mutagenizing agent, b) growing the at least one poppy seed to produce a plant bearing a leaf or an immature poppy capsule, optionally through multiple self-fertilized generations, c) sampling the leaf or poppy capsule for the presence of thebaine, oripavine, morphine and codeine, and d) repeating steps a) to c) until a poppy plant of Papaver somniferum is obtained having thebaine and oripavine constituting about 50% by weight or greater of the alkaloid combination consisting of morphine, codeine, thebaine and oripavine
http://www.freepatentsonline.com/y2002/0106761.html

Papaver Somniferum Strain With High Concentration Of Thebaine
JOHNSON & JOHNSON 2009
The present invention is directed to the improved production of thebaine. More particularly, the present invention relates to the use of a mutagenized Papaver somniferum poppy plant to produce thebaine in higher yield.
http://www.freepatentsonline.com/y2009/0227796.html

`Dhawal`, a high alkaloid producing periwinkle plant
http://www.patentstorm.us/patents/6548746/description.html

ect, ect you could go on all day citing examples of chemically induced mutagenesis to produce high alkaloid levels or novel alkaloids ect. Be aware that the technique is still used extensively to this day! This is generally how they discover the plants with desirable traits, then profile its DNA. And can then figure out from those novel poppies DNA what alleles are responsible for a certain alkaloids biosynthesis when comparing it to a normal poppy DNA.  

Horizontal Gene Transfer
Now scientists discovered at some stage (?) that Agrobacterium as well as certain yeasts, fungi, bacterium and virii were capable of Horizontal gene transfer;

"Horizontal gene transfer (HGT), also Lateral gene transfer (LGT), is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism. By contrast, vertical transfer occurs when an organism receives genetic material from its ancestor, e.g. its parent or a species from which it evolved.
Most thinking in genetics has focused upon vertical transfer, but there is a growing awareness that horizontal gene transfer is a highly significant phenomenon, and amongst single-celled organisms perhaps the dominant form of genetic transfer. Artificial horizontal gene transfer is a form of genetic engineering."
http://en.wikipedia.org/wiki/Horizontal_gene_transfer

Agrobacterium was found to have, as you stated a type of DNA. Called a plasmid it is not good to think of this like normal DNA, as it is more like a package made by the bacteria containing everything it needs to replicate independently without any more help from the bacterium that made it.

"Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. "

 Illustration of a bacterium with plasmid enclosed showing chromosomal DNA and plasmids.
http://en.wikipedia.org/wiki/Plasmid

During horizontal gene transfer (if a plant has been infected with agrobacterium and it is inside the plant), the bacterium can conjugate with the plants cells in which case the bacterium would be the donor and the plant cell a recipient;

As you can see the plasmid does not hop over to the recipient cell, rather replicates itself in there.

"The plasmid T-DNA is integrated semi-randomly into the genome of the host cell (Francis and Spiker, 2005. Plant Journal. 41(3): 464.), and the virulence (vir) genes on the T-DNA are expressed, causing the formation of a gall. The T-DNA carries genes for the biosynthetic enzymes for the production of unusual amino acids, typically octopine or nopaline. It also carries genes for the biosynthesis of the plant hormones, auxin and cytokinins. By altering the hormone balance in the plant cell, the division of those cells cannot be controlled by the plant, and tumors form. The ratio of auxin to cytokinin produced by the tumor genes determines the morphology of the tumor (root-like, disorganized or shoot-like)."
http://en.wikipedia.org/wiki/Agrobacterium

So it actually semi-randomly changes the plants genome, therefore changing the plants chromosomal DNA which is what is passes on to its offspring. The T-DNA (transfer DNA);

"The transfer DNA (abbreviated T-DNA) is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes. It derives its name from the fact that the bacterium transfers this DNA fragment into the host plant's nuclear DNA genome. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the left border and terminated at the right border and requires the vir genes of the Ti plasmid.

The bacterial T-DNA is about 20,000 base pairs long and contains genes that code for enzymes synthesizing opines and phytohormones. By transferring the T-DNA into the plant genome, the bacterium essentially reprograms the plant cells to grow into a tumor and produce a unique food source for the bacteria. The synthesis of the plant hormones auxin and cytokinin enables the plant cell to grow uncontrollably, thus forming the crown gall tumors typically induced by Agrobacterium infection. The opines are amino acid derivatives used by the bacterium as a source of carbon and energy."

So at the end of the day, agrobacterium are very handy for GM applications. But straight out of the soil there are programmed to induce root tumors for example in A. rhizogenes, and nothing else!

Recombinant DNA

"Agrobacterium-mediated T-DNA transfer is widely used as a tool in biotechnology. In genetic engineering, the tumor-promoting and opine-synthesis genes are removed from the T-DNA and replaced with a gene of interest and/or a selection marker, this is required so that it is possible to establish which plants have been successfully transformed. Examples of selection markers include neomycin phosphotransferase, hygromycin B phosphotransferase (which both phosphorylate antibiotics) and phosphinothricin acetyltransferase (which acetylates and deactivates phosphinothricin, a potent inhibitor of glutamine synthetase). Agrobacterium is then used as a vector to transfer the engineered T-DNA into the plant cells where it integrates into the plant genome. This method can be used to generate transgenic plants carrying a foreign gene."

And another 'ase' for them Codeinone Reductase Protein! But as you can see the tumor promoting and opine-synthesis genes are removed from the T-DNA of the plasmid, they call this an unarmed plasmid now. Digging straight out of the soil our bacteriums plasmids would be armed to the teeth. We could aquire unarmed plasmids, but would we be able to remove the genes of interest from the target DNA and then insert them into a plasmid followed by annealing as in the diagram below?

"Recombinant DNA (rDNA) is a form of DNA that does not exist naturally, but is created by combining DNA sequences that would not normally occur together.[1] In terms of genetic modification, recombinant DNA is introduced through the addition of relevant DNA into an existing organismal DNA, such as the plasmids of bacteria, to code for or alter different traits for a specific purpose, such as antibiotic resistance.[1] It differs from genetic recombination in that it does not occur through processes within the cell, but is engineered.[1] A recombinant protein is a protein that is derived from recombinant DNA.[2]"


A simple example of how a desired gene is inserted into a plasmid. In this example, the gene specified in the white color becomes useless as the new gene is added


http://en.wikipedia.org/wiki/Recombinant_DNA

I highlighted the definition of recombinant protein, which is just a protein that is made using the r-DNA to guide it, so if the r-DNA has been changed. The subsequent recombinant proteins that are made will be changed accordingly. And is used in one of the quotes coming up, there are other types of recombinant DNA like chimeric plasmids that add additional strands to the DNA (not change it) but for what we are talking about.....

Codeinone reductase from alkaloid poppy
Johnson & Johnson Research Pty. Limited (AU)   

At least 10 codeinone reductase alleles are present in the genome of the poppy Papaver somniferum . Isolation, characterization and functional expression of four of the 10 genes encoding codeinone reductase as described herewith enables methods for controlling alkaloid production in opium poppy plants and cultures by providing a target for genetic manipulation.

(after Isolation, characterization and functional expression of some of the genes from above. So a whole lot of tedious lab work later.....)

Transformation of Plants with Nucleotide Sequences from Genes Encoding Codeinone Reductase Proteins
Plant Materials

Two plant lines were used in transformation experiments. These were Nicotiana tabacum line Wisconsin38, and Papaver somniferum line C048. Preparation of plant materials and tissue culture and transformation conditions were as described in An et. al (1986), Hooykaas and Schilperoort (1992) and PCT Application PCT/AU99/00004, all of which are incorporated herein by reference.

Bacterial Strains and Vectors

The disarmed Agrobacterium tumefaciens strain LBA4404 was used in transformation experiments. DNA constructs capable of expressing the codeinone reductase genes were prepared in a binary vector containing a 35 S-nptII selectable marker, and transformed into the N. tabacum and P. somniferum lines.

Successful transformation of these plant lines was achieved as judged by

(a) regeneration of N. tabacum plants on medium containing 100 mg/l kanamycin indicating expression of the nptII selectable marker, which was verified by NPTII enzyme assays. Coexpression of the codeinone reductase gene was determined by RT-PCR (reverse transcriptase polymerase chain reaction) assay.
(b) successful selection of transformed cell cultures of P. somniferum using the same nptII selectable marker indicative of expression from the vector, followed by the generation of typeI and typeII embroyogenic callus prior to the production of transformed plants.

Thus, the identification and cloning of genes for codeinone reductase from P. somniferum now provides a means by which alteration of the enzymatic step(s) involving this can be achieved. The overexpression of these sequences can be achieved using vectors which express one or more of the codeinone reductase alleles, while downregulation of general codeinone reductase activity or the activity of specific alleles can be achieved using vectors expressing antisense, ribozymes, plus-sense cosuppression or RNAi sequences from regions conserved between the codeinone reductase alleles or other sequences which are unique to each allele. These genes encoding the sense, antisense, ribozyme, RNAi or other such sequences can be delivered as transgenes stably integrated into the poppy genome or transiently in the form of a viral vector.

Although the invention has been described with reference to specific embodiments, modifications that are within the knowledge of those skilled in the art are also contemplated as being within the scope of the present invention.
http://www.freepatentsonline.com/7193127.html




note; Just a question? Johnson and Johnson, in Australia is a well known brand name. It has a very good image associated with baby products, and a very family friendly image. Here is there website if you not familiar; http://www.jnjaust.com.au/
I was a little taken back to find out they were the people doing most of the (successful) work with poppy alkaloids, and all the patents I linked in this post were theirs! Tasmania Alkaloids who produces all the poppies in Tasmania has some poppy straw processing patents, but would have to pay Johnson and Johnson for their genetics. J and J would be making billions off Oxy- drugs and all the morphs produced each year down there! Maybe Tas Alk is just a subsidiary of J and J and they run the whole show ??? But do not want to damage their reputation as being nice guys not drug dealers! If their was no under the counter oxy-contin ect ect hill billy herion abuse I would not call it like that (but their is, millions of $$$ worth per annum).
« Last Edit: May 14, 2010, 12:45:23 AM by Naf1 »

Naf1

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Re: Genetic Modification at home
« Reply #2 on: May 14, 2010, 01:30:09 AM »
NOTE; I just clicked on some of those original links and discovered hairy root culture, ;D oops! I was talking about GM via agrobacterium.I will not delete the above post in case anyone is interested in the basics! But in a lot of cases (like the jellyfish) the Ri plasmid is has been modified as above then the roots damaged to let it in. In some cases like the one linked below they give the target plant hairy root as that is now transgenic and contains the plants genes plus agrobacterium genes, so the vector system is introduced into the transgenic roots allowing the bacterium to enter more easily than it would with healthy 100% plant genes.

If they are talking about about A.rhizogens vector systems, there are talking about the methods described above, to introduce genes into medicinal plants via plasmids. The hairy root culture with unmodified A rhizogens, is used for producing secondary metabolites that are normally biosynthesized in the roots! So saffy is all good, calamus....

Hairy Root and Its Application in Plant Genetic Engineering

Hairy roots grow rapidly, show plagiotropic growth, and
are highly branched on phytohormone-free medium. The transformed
root is highly differentiated and can cause stable and
extensive production of secondary metabolites, whereas other
plant cell cultures have a strong tendency to be genetically and
biochemically unstable and often synthesize very low levels of
useful secondary metabolites
(Rhodes et al. 1990; Merkli et al.
1997; Kittipongpatana et al. 1998).
Most importantly, A. rhizogenes
can transfer T-DNA from binary vectors and enable the production
of transgenic plants containing foreign genes carried on a
second plasmid.

http://www.shutcm.com/shutcm/zybzhyjzx/xswz/swjswz/images/2006/10/13/993.pdf
« Last Edit: May 14, 2010, 01:52:14 AM by Naf1 »

Sedit

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Re: Genetic Modification at home
« Reply #3 on: May 14, 2010, 04:50:34 AM »
Thank you for the well written response Naf.

I am happy to learn that the DNA is incorperated thru the Pilius(Bacteria Dick HA!!) and not the result of a dead lysed bacteria like I had thought before.

I am fairly confident that modification of a root can be done with relative ease but what of synthesizing Plasmids. Its something I have not really researched yet since I have not reached that level of understanding yet but I do understand some bits about gene isolation and I would think that plasmid synthesis involves some enzyme to link the terminal ends of the isolated gene.
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Re: Genetic Modification at home
« Reply #4 on: May 14, 2010, 06:24:39 AM »
Vesp was talking about buying the bacterium, having a lab prepare it and clone it.

But otherwise you first need the correct type of Restriction Enzyme, then the chunk of compatible DNA, and DNA ligase.

"A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded or single stranded DNA at specific recognition nucleotide sequences known as restriction sites."

They are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments. For optimal use, plasmids that are commonly used for gene cloning are modified to include a short polylinker sequence (called the multiple cloning site, or MCS) rich in restriction enzyme recognition sequences. This allows flexibility when inserting gene fragments into the plasmid vector; restriction sites contained naturally within genes influence the choice of endonuclease for digesting the DNA since it is necessary to avoid restriction of wanted DNA while intentionally cutting the ends of the DNA. To clone a gene fragment into a vector, both plasmid DNA and gene insert are typically cut with the same restriction enzymes, and then glued together with the assistance of an enzyme known as a DNA ligase.[27][28]"

Next quote (from dna ligase quote)

DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.
One vital, and often tricky, aspect to performing successful recombination experiments involving the ligation of cohesive-ended fragments is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4) which is most active at 25°C. However in order to perform successful ligations with cohesive-ended fragments ("sticky ends"), the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated.[1] If the ambient temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding.[citation needed] The shorter the DNA fragments, the lower the Tm.
Since blunt-ended DNA fragments have no cohesive ends to anneal, controlling the optimal temperature becomes much less important. The most efficient ligation temperature will be the temperature at which T4 DNA ligase functions optimally (T4 DNA ligase is the only commercially-available DNA ligase to anneal blunt ends).[1] Therefore, the majority of blunt-ended ligations are carried out at 20-25°C.
The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli and other bacteria."

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

So if one can get the correct restriction enzyme and DNA ligase it is not impossible, the trickiest part being identification and isolation of the correct chunk of DNA and actually being able to cut that correctly. Sound impossible not really using DNA electrophoresis, and gel electrophoresis kits are sold widely to interested people and tertiary school people and teachers ect. Using enzymes or more of those DNA restriction endonuclease the poppy (for example) DNA loses it backbone and the gel separation takes effect separating the DNA into corresponding fragments. The required fragments are collected and used above! http://bcs.whfreeman.com/thelifewire/content/chp16/1602001.html

Hard in theory (especially if you get right into it) but if you can get the enzymes, ligase ect in question. It is not that hard a procedure, similar to what you have set up now gut maybe have to monitor heat and keep at 25*C  little things like that.

 

« Last Edit: May 14, 2010, 06:26:59 AM by Naf1 »

Naf1

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Re: Genetic Modification at home
« Reply #5 on: May 14, 2010, 06:39:20 AM »
And because it is so easily done once understood and access to the above is obtained, this is the outcome;

Illegal GM rice widely available in Hubei
http://www.gmwatch.org/latest-listing/1-news-items/12124-illegal-gm-rice-widely-available-in-hubei-

Illegal GM flax seed found in Marks & Spencer bread
http://www.anh-europe.org/news/illegal-gm-flax-seed-found-in-marks-spencer-bread

IRELAND'S GENETICALLY MODIFIED FOOD SCANDAL
http://www.gmfreeireland.org/pakrac/index.php

Pro-Biotech farmer plants illegal GM seeds
http://www.lifeinitaly.com/node/26815

and many many more examples can be found, and it is still relatively early days!

Sedit

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Re: Genetic Modification at home
« Reply #6 on: May 18, 2010, 04:31:42 AM »
To cool not to share. Something I have thought of for a while put into practice. Dremel is the only thing I could think of and appearently this fellow also that can spin at the speeds needed but I always woundered to to link the rotor safely with the test tubes themself. He uses a piece of starboard it looks like and shapes that into a holder for his test tubes. A must have for anyone getting serious into DIY Genetic modification.

http://diybio.org/2009/12/30/diy-centrifuge-using-dremel-tool/



Also on a side note. A quick google will find you a new project that bacteria that eats explosives was fitted with glowing genes to spread over a field of suspected landmines. Within an hour they colonize and cause every area with landmines under it to glow a bright green color at night which is enhanced with the use of a blacklight.
There once were some bees and you took all there stuff!
You pissed off the wasp now enough is enough!!!

Vesp

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Re: Genetic Modification at home
« Reply #7 on: May 18, 2010, 05:09:47 AM »
That is very interesting, where is it the dremel attachment can be obtained for a cheaper price? That is a lot of money for a plastic circle... Might be able to easily make one out of wood, if you have a dremel already :p
 If this doesn't work, one might also be able to get a full up one at a university surplus for cheaper.

http://citsci.blogspot.com/2009/11/centrifuge-revisited.html There is also this dangerous looking device. I like the dremel better.
« Last Edit: May 18, 2010, 05:12:34 AM by Vesp »
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Naf1

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Re: Genetic Modification at home
« Reply #8 on: May 29, 2010, 11:56:23 PM »
Also regarding my J&J comments above;


Johnson & Johnson Pharmaceutical Research and Development (J&JPRD) is a subsidiary of Johnson & Johnson that is responsible for discovering and developing pharmaceutical drugs. J&JPRD has research sites located in Raritan, New Jersey, Spring House, Pennsylvania, La Jolla, California, Beerse, Belgium and Toledo, Spain.
J&JPRD was created through the merge of various research organizations including McNeil Pharmaceuticals, Janssen Research Foundation, Three Dimensional Pharmaceuticals and the R. W. Johnson Pharmaceutical Research Institute.
http://en.wikipedia.org/wiki/Johnson_&_Johnson_Pharmaceutical_Research_and_Development

btw; That dremelfuge is a great idea! The link to the shop did not work for me, so could not see how much he wanted, but in regards to making your own (of course you could). You could make out of wood, plastic (use the dremel to make the pastic bit) or a single use one out of duct tape if you wanted!
« Last Edit: May 30, 2010, 12:04:25 AM by Naf1 »

Naf1

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Re: Genetic Modification at home
« Reply #9 on: May 30, 2010, 05:26:57 AM »
Prenylation of olivetolate by a hemp transferase yields cannabigerolic acid, the precursor of tetrahydrocannabinol
Monika Fellermeier, Meinhart H. Zenk
FEBS Letters 427 (1998) 283^285

Abstract A new enzyme, geranylpyrophosphate:olivetolate geranyltransferase (GOT), the first enzyme in the biosynthesis of cannabinoids could be detected in extracts of young leaves of Cannabis sativa. The enzyme accepts geranylpyrophosphate (GPP) and to a lesser degree also nerylpyrophosphate (NPP) as a cosubstrate. It is, however, specific for olivetolic acid; its decarboxylation product olivetol is inactive as a prenyl acceptor.

http://www.druglibrary.org/crl/receptors/agonists/Fellermeier%20et.al%2098%20Prenylation%20FEBSLetts.pdf

The Gene Controlling Marijuana Psychoactivity
MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF ?1-TETRAHYDROCANNABINOLIC ACID SYNTHASE FROM CANNABIS SATIVA L.

Supaart Sirikantaramas, Satoshi Morimoto, Yoshinari Shoyama, Yu Ishikawa, Yoshiko Wada, Yukihiro Shoyama and Futoshi Taura
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of September 17, pp. 39767–39774, 2004

Abstract

?1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic acid into THCA, the precursor of ?1-tetrahydrocannabinol. We cloned a novel cDNA (GenBank™ accession number AB057805) encoding THCA synthase by reverse transcription and polymerase chain reactions from rapidly expanding leaves of Cannabis sativa. This gene consists of a 1635-nucleotide open reading frame, encoding a 545-amino acid polypeptide of which the first 28 amino acid residues constitute the signal peptide. The predicted molecular weight of the 517-amino acid mature polypeptide is 58,597 Da. Interestingly, the deduced amino acid sequence exhibited high homology to berberine bridge enzyme from Eschscholtzia californica, which is involved in alkaloid biosynthesis. The liquid culture of transgenic tobacco hairy roots harboring the cDNA produced THCA upon feeding of cannabigerolic acid, demonstrating unequivocally that this gene encodes an active THCA synthase. Overexpression of the recombinant THCA synthase was achieved using a baculovirus-insect expression system. The purified recombinant enzyme contained covalently attached FAD cofactor at a molar ratio of FAD to protein of 1:1. The mutant enzyme constructed by changing His-114 of the wild-type enzyme to Ala-114 exhibited neither absorption characteristics of flavoproteins nor THCA synthase activity. Thus, we concluded that the FAD binding residue is His-114 and that the THCA synthase reaction is FAD-dependent. This is the first report on molecular characterization of an enzyme specific to cannabinoid biosynthesis.

http://www.jbc.org/content/279/38/39767.abstract

"Moreover, we attempted the expression of the active
THCA synthase in tobacco hairy roots. Interestingly, the transformed
tobacco hairy roots produced THCA on exogenous addition
of the biosynthetic precursor CBGA. We also describe the
development of THCA-producing tobacco."

That tobacco is somewhat novel, but using cannabis and regulating the expression of such proteins as GOT and THCA as the aforementioned patent describes for papaver sonmiferum could be a very worthwhile venture! You can see what you need to go through in the above paper, the experimental has the procedures.

Crystallization of ?1-tetrahydrocannabinolic acid (THCA) synthase from Cannabis sativa
Yoshinari Shoyama,a Ayako Takeuchi,a Futoshi Taura,a Taro Tamada,b Motoyasu Adachi,b Ryota Kuroki,b Yukihiro Shoyama,a and Satoshi Morimotoa
Acta Cryst. (2005). F61, 799–801

Delta-1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure–function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A ° resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A ° . The calculated Matthews coefficient was approximately 4.1 or 2.0 A ° 3 Da1 assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1952348/pdf/f-61-00799.pdf
« Last Edit: May 30, 2010, 05:32:20 AM by Naf1 »

Vesp

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Re: Genetic Modification at home
« Reply #10 on: October 11, 2010, 01:48:29 AM »
I figured I might as well post this idea somewhere so it can be commented on, and more importantly so it isn't lost for later on down the road when genetic engineering becomes DIY and home biology really takes off..

It is my understanding that since fungi are more simple than plants and animals they can handle more genetic manipulations. It is also my understanding and experience that they are easier to grow.

This leads me to believe that fungi are probably easier to add new DNA sequences too and produce new alkaloids, etc.

Considering what we know about endophytes, which are fungi that live in plants and some produce useful quantities of alkaloids, it might be worth finding an endophyte that is extremely fast growing, handles cold and hot weather when living in the plant and is easy to grow in a submerged culture. Than genetically modify it to produce various substaces such as safrole, DMT, Psilocybin, Mescaline, cocaine, and so on.
Whatever plant it lives in would  also contain the chemicals and could be distributed effectively by means of seeds. The plants could than be grown out for extraction, or the fungi could be isolated and grown out for a more convenient production.

Again, obviously this is a long ways away, but I think endophytes are a good target to modify, since they have a duality of producing chemicals of interests both with in a plant and in a culture - as well as being more easily modified and easier and faster  strain isolation than with plants.
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Vesp

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Re: Genetic Modification at home
« Reply #11 on: December 02, 2010, 01:47:40 PM »
Has anyone done any additional research into this topic, and if so - find anything new and interesting?
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Helgoland

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Re: Genetic Modification at home
« Reply #12 on: February 21, 2011, 05:58:34 PM »
Sorry if I'm reviving an old thread, but: Phosphate!
http://en.wikipedia.org/wiki/Transfection
Should be cheap and not overly difficult. Perhaps soon bees will be growing their drugs in lawn form. (Old fantasy of mine.)
Also: doi:10.1016/0042-6822(73)90341-3
Anyone interested?

Helgoland

Vesp

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Re: Genetic Modification at home
« Reply #13 on: February 21, 2011, 06:15:28 PM »
Nothing wrong with reviving an old thread! In fact its better than starting a new one of the same topic.

Quote
One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973[6] (see also [7]). HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it, the DNA.


Quote
Perhaps soon bees will be growing their drugs in lawn form. (Old fantasy of mine.)

Same here!


Now is this for animal cells only or does it also work with plant cells? It seems to me that if it is to work on plant cells, you will need to remove the cell wall composted of cellulose in order for it to work?

Bacterial cells might be a better option, or possibly fungal cells (which are composed of chitin, which is to some extent similar to cellulose and may lead to the same issues, if there are any)
Either way, both can be stored and grown in large vats.

It seems to me the real problem here is not actually getting an organism to take up the DNA, but actually getting the right segments of DNA, which means isolating it via something like electrophoresis, multiplying it via PCR. After that, I believe it should be pretty easy - I think many bacterial cells will even absorb DNA due to a temperature shock, such as E. Coli - but I could be wrong.
Another task is also the enzymes needed - such ones as restriction enzymes... and possibly things like cellulose/chintinase enzymes if one needs to remove cell walls in order for success.

Also if one is to use something such as agrobacterium rhizogenes to insert a gene into a plant, that needs to be acquired.

A. rhizogenes or the A. tumefacians  might be considered a plant pathogen and would thus be regulated.


Edit:

http://www.molecular-plant-biotechnology.info/gene-transfer-methods-in-plants/calcium-phosphate-precipitation-method-for-gene-transfer.htm


Quote
Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.
-- http://www.biotechnology4u.com/plant_biotechnology_gene_transfermethods_plants.html


Good stuff.

I wish we could make some real progress with this - and especially with endophytes - which are either bacteria or fungi that live within a variety of plants. This allows for them to be transfered easily from place to place in the form of a plant or a seed, grow in nature in the lawn - but also be isolating and grown in a liquid culture or on agar - allowing for such compounds to be produced in a variety of ways, and increase the ease at which other people can isolate and mutate them to produce, possibly better yielding strains.

The Acremonium fungi is a good candidate to try to turn into a producer of interesting alkaloids, as it is rumored to already produce many different ones (ergot, nicotine, etc...) and is available to most who can identify toxic fescue (its host)
« Last Edit: February 21, 2011, 06:22:13 PM by Vesp »
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Helgoland

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Re: Genetic Modification at home
« Reply #14 on: February 21, 2011, 06:20:52 PM »
http://www.molecular-plant-biotechnology.info/transgenic-animals/calcium-phosphate-precipitation.htm
Somewhere I read it could also be done with plants, but there were some issues with plant walls.
A callus culture, maybe?
And about getting the right bit of DNA-brute force could be your friend if no other approach is found.
Any papers/experimental procedures?

Vesp

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Re: Genetic Modification at home
« Reply #15 on: February 21, 2011, 06:28:45 PM »
Problem with brute force is that, even if you do get a plant cell that is able to produce morphine - how are you going to isolate it from the others that are expressing other traits, or it may uptake other DNA that is harmful to it...

You would literally have to grow each mutant cell into a new plant and test for alkaloid presents, it seems?

Generally what is done, is the wanted DNA is isolated, grown out, and is conntected to a bit of DNA that is resistant to a biocide (generally in a plasmid, when agrobacterium is used) - after the cells are grown out for a while, the biocide is added, and all of the cells that did not take in the DNA are killed, than you are left with one that is resistant to the biocide, and has the alkaloid producing gene...
Not to sure if that would also be done with the calcium phosphate method.

Unless you have some info on brute force attempts/success?

Edit: I think brute force would be fantastic for doing mushrooms - combine genetics of oyster, shiitake, and other edibles together to yeild new edible mushrooms that you have patented OR add biolumiscent genes from P. stripticus.
Someone ought to grow out P. stripticus, extract its DNA, and do the Calcium phosphate treatment with that DNA on a non-bioluminescent fungi - it would be a super easy test to see if you've succeeded at DIY at home genetic engineering.

I may try this..I already have some bioluminescent fungi, but the sad reality is I'm pretty busy and probably won't get around to it.
I would also probably need to cut up the DNA a bit?
« Last Edit: February 23, 2011, 02:44:16 AM by Vesp »
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frronkis

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Re: Genetic Modification at home
« Reply #16 on: February 26, 2011, 01:28:49 AM »
Hi guys...I've also been researching the use of genetic modification, particularly of plants, to produce medicinally-useful compounds. To this effect, my own research has focused on somatic fusion (protoplast fusion):

(From wikipedia http://en.wikipedia.org/wiki/Somatic_fusion)

"Somatic fusion, also called protoplast fusion, is a type of genetic modification in plants by which two distinct species of plants are fused together to form a new hybrid plant with the characteristics of both, a somatic hybrid. Hybrids have been produced either between the different varieties of the same species (e.g. between non-flowering potato plants and flowering potato plants) or between two different species (e.g. between wheat triticum and rye secale to produce Triticale)."

Somatic fusion is actually pretty easy as far as genetic modifications go, and can be applied to both plant and animal cells.
(From wikipedia, con't)

"The somatic fusion process occurs in four steps:[2]

   1. The removal of the cell wall of one cell of each type of plant using cellulase enzyme to produce a somatic cell called a protoplast
   2. The cells are then fused using either an electric shock (electrofusion) to join the cells and the nuclei fused together or by chemical treatment(usually polyethyelene glycol). The resulting fused nucleus is called heterokaryon.
   3. The somatic hybrid cell then has its cell wall induced to form using hormones

   4. The cells are then grown into calluses which then are further grown to plantlets and finally to a full plant, known as a somatic hybrid."

Some of the benefits of somatic hybridization:

"Characteristics of Somatic Hybridization and Cybridization

1. Somatic cell fusion appears to be the only means through which two different parental genomes can be recombined among plants that cannot reproduce sexually (asexual or sterile).

2. Protoplasts of sexually sterile (haploid, triploid, and aneuploid) plants can be fused to produce fertile diploids and polyploids.

3. Somatic cell fusion overcomes sexual incompatibility barriers. In some cases somatic hybrids between two incompatible plants have also found application in industry or agriculture.

4. Somatic cell fusion is useful in the study of cytoplasmic genes and their activities and this information can be applied in plant breeding experiments."

So, somatic fusion represents another potential avenue for crossing unrelated plant species to produce novel, alkaloid-bearing combinations. Check out the pdf for details.

Some highlights from the pdf for the lazy:

"Protoplast fusion also enables the genetic manipulation of vegetatively propagated crops, such as sterile or subfertile
individuals, and those plants, including woody species, with naturally long life cycles.
"

"The ability to isolate protoplasts that, when cultured under defined conditions, divide mitotically and regenerate plants has now been established for many species, including woody plants."

"Induced protoplast fusion can be achieved using chemical and electrical treatments. In both cases, fusion is a two-stage process. First, protoplasts are brought into close membrane contact, the degree of plasma membrane adhesion depending on the parental protoplasts. Tight contact may occur only in localized regions between adhering protoplasts. Subsequently, the plasma membranes are stimulated to interact, for example, by modification of the electrical charges on the membranes, resulting in protoplast fusion."

"The extent of protoplast fusion, heterokaryon formation, and survival of fusion products can be monitored using naturally occurring visual markers."



« Last Edit: February 26, 2011, 01:38:31 AM by frronkis »

Vesp

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Re: Genetic Modification at home
« Reply #17 on: March 03, 2011, 03:08:44 AM »
DNA fragmentation would be wanted for the Calcium Phosphate transfection, it sounds like DNA Fragmentation can be caused by heat,
http://www.kreatech.com/Default.aspx?tabid=119 and a few other things - looks fairly simple...

but might be still problematic because you will need promoter codes, etc.. http://cls.casa.colostate.edu/transgeniccrops/how.html
« Last Edit: March 03, 2011, 03:12:17 AM by Vesp »
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solidstone

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Re: Genetic Modification at home
« Reply #18 on: March 03, 2011, 04:41:06 AM »
See attached pdf.
recently did this experiment and it seems adaptable to the home setting.  Step by step procedure which makes it nice for learning purposes.  The procedure is for a plasmid transformation of e. coli.

note:this company looks as it could be of some use for 'learning purposes'

also it appears there are a lot of plasmid purification business... there not terribly cheap, but if you know what you want it could easily be worth the chunk of change they require.
« Last Edit: March 03, 2011, 04:44:59 AM by solidstone »

timecube

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Re: Genetic Modification at home
« Reply #19 on: March 03, 2011, 01:19:15 PM »
This may have been posted somewhere else, but I didn't see it in the above references.

http://www.sciencenews.org/view/generic/id/57257/title/Chemists_pin_down_poppys_tricks_for_making_morphine

(official paper link, haven't checked around yet for a full copy)
http://www.nature.com/nchembio/journal/v6/n4/abs/nchembio.317.html

The genes themselves are particular interesting, and the article (and presumably the full paper) give a run down of how they were discovered.

Oddly the article's commenters are particularly dense to be readers of something called Science News.