Hey! Yesterday u/mjauderp posted something interesting on reddit. asked why he did not post that on here he said



So, here is the text from the pastebin link he posted.

Biosynthesis of DMT with modified e.coli

=== Introduction ===

I want to begin with writing that it is never impossible to do anything, if one just have the curiosity and stamina needed to keep pushing the limits.

I smoked DMT that a friend gave me. Then I realized how difficult it was to obtain more of it - but that it was possible to easily extract from plants. Wishing to manufacture more of it at a large scale, I researched. It turned out that it would be quite difficult to grow plants containing DMT. Mimosa Hostilis is a tree! Other plants do not have the content percentage worth for growing. So I put that idea in the trashbin.

A year passed. I studied some microbiology and saw that biosynthesis of DMT only requires two enzymes. Bioluminescence, the equivalent of "Hello World" in the world of biohacking, is about as complex.

So I decided is was time to reanimate this old corpse of an idea of mine, and write the code.

Somehow all my great ideas and plans for my life are totally illegal.

=== Links ===
Get the very basics: https://en.wikipedia.org...gma_of_molecular_biology
Coding for proteins: https://en.wikipedia.org...tic_code#DNA_codon_table
The FASTA-format, used to describe proteins and other: https://en.wikipedia.org/wiki/FASTA_format
Standard template genes: http://parts.igem.org/Catalog
For help with understanding terminator genes: http://www.entelechon.co...rna-secondary-structure/
Genome and metabolic pathways of E. coli (IMPORTANT!): http://ecocyc.org/ and http://biocyc.org/

Online shops selling synthetic DNA:
* http://www.plasmid.com/
* http://www.genscript.com/gene_synthesis.html
* http://www.genewiz.com/public/gene-synthesis.aspx
* Google

Unrelated about THC: http://www.cannabis-med....bis_artikel.php?id=329#1

=== E. Coli ===
Escherichia coli is a procaryotic life-form that lives in your body. It is the standard organism for biological experiments, and as such it is one of the most studied organisms around. Its genome and its metabolic network is fully expolored, and available at the internet.
Procaryotic life have no nuclei or any other "body parts". Procaryotes are literary just bags of chemicals. This means that chemistry and genetics are a bit easier to understand, than for eucaryotes.

=== Biosynthesis of DMT ===
The basic idea is to extend the metabolic pathway of e coli by inserting new genes into it. The genes code for proteins that make it happen (exactly like described in https://en.wikipedia.org...ltryptamine#Biosynthesis). It is possible to buy synthetic genes on plasmids from the internet for a few hundred dollars. Plasmids are circular strings of DNA that, when bought from synthesizers also often encode for resistance against some form of antibiotics, such as amplifilin. The idea is that after you have inserted the plasmid into a colony of E. coli, you add the antibiotic specific to the plasmid in order to kill off all individual organisms that did not absorb the plasmid. Insertion of genes with the help of a plasmid vector is very well described and can be done in a kitchen. There are youtube tutorials. For example, https://www.youtube.com/watch?v=slY4qrnZIM8.

L-Tryptophan, one of the 22 essential amino-acids (which exist in all living bodies), is transformed into DMT through interaction with two enzymes: AAAD (Aromatic L-amino acid decarboxylase) and INMT (Amine N-methyltransferase).

E. coli lacks both these enzymes.

These enzymes consume co-enzymes that are available in the e.coli.

(You can look up the available chemicals in e coli and their relationship with its genome by using ecocyc.org or biocyc.org.)

If E.coli contained AAAD and INMT, they would interact with other chemicals present in the cell and produce new chemicals. Going through the list of all chemicals that can interact with the two enzymes, I found that AAAD would produce the phenetylamine and tyramine as a by-product. This is important, as ingesting tyramine in combination with a mono-amine oxidase inhibitor (MAOI) could cause hypertensive crisis. This could be lethal to a psychedelic user ingesting the product.

=== Genetics ===
I took the code for the enzymes from uniprot.org and compiled them into the DNA. For procaryotes this means that one simply translated each amino-acid into codons (DNA-basepair triplets) that code for them.

The other parts it took from the biobricks project: An annual competition held by universities to teach students to genetically modify organisms into doing various mostly useful things. (http://parts.igem.org/Catalog?title=Catalog) The biobricks project is also quite a useful place to start off ones own studies, its very pedagogical.

The central dogma of genetics is that DNA is transcribed into RNA, that is then translated into sequences of amino-acids. These sequences are proteins, and enzymes are proteins.

promoter


The promoter recruits transcriptional machinery, that attach to the DNA and transcribes it into mRNA.

The transcription strength is perhaps one of the most important knobs one could experiment with. Too high transcription would result in too much of the organisms metabolic system being kidnapped for the manufacture of enzymes, which would slow growth. A too low transcription would result in an organism that does not produce enough amounts of DMT.

Ribosome Binding Site (RBS)


The RBS binds the mRNA to ribosomes, that translates the mRNA into amino acids.

The rest of the gene below, with the exception of the terminator, is code that will be translated into enzymes. Both enzymes start with methodine, which is also a start-codon. This means that no "cutting and pasting" is needed. Between the code that translates to the enzymes, there are stop codons and a spacer that does not initiate translation.

I got the code for the enzymes from uniprot.org. They are both from the Mus Muscus species of mice (the common house mouse.)

The INMT-enzyme in FASTA-format.


The same enzyme encoded into DNA looks like this:



Remember, the DNA above is two steps away from the translation done by the ribosome. DNA is transcribed into RNA by DNA transcriptase, RNA is translated into proteins by ribosomes. But we are interested in the DNA that we can put into the organism, so that is why I worked my way backwards to DNA.



Makes the ribosome stop translation. The ribosome continues its travel down the single-helix RNA though, so it will encounter the next enzyme-encoding sequence as well.



Does nothing at all, just to separate the proteins in space, and give the ribosome a chance to properly emit the INMT before beginning on the AAAD.

AAAD-enzyme in FASTA-format.


Below is the DNA sequence that codes for the protein above.





Terminates translation and makes the ribosome emit the protein.



Terminator that forms a physical loop out of the single-helix RNA equivalent. The loop is formed by the palindrome contained within the sequence. Read more about it at biobricks wiki under the topic terminators.

The whole sequence looks like this:



=== Notes ===
I have not checked the above gene for palindromes. There are multiple ways of encoding most amino acids, I always used the same encoding for each amino acid. It might work, it might not work.

In theory, this is what I or you could do:
1) Buy the gene above on a plasmid from some online shop
2) Introduce the plasmid into a population of e coli (see youtube tutorials)
3) Kill off the e coli that did not assimilate the plasmid using a pencilin
4) Grow
5) Dry the biomass and harvest it using straight to base, or similiar

THIS IS PURELY THEORETICAL. I have no idea if it works. Please contribue with more ideas.

One does not begin designing synthetic biological lifeforms after "studying some molecular biology for a year."

It all looks really easy in theory, but let me tell you, as someone who has spent a LOT of time working in various labs at various institutions, it is NEVER this easy. Oh, it might look easy, but next thing you know, you're in the lab for your 72nd consecutive hour, wondering if you should give up your degree and open a gas station in the Philippines or something...

Sorry, that got personal.

In all seriousness though, I have my doubts. That said - it only really has to work once: if the new code is stable, you can propagate and distribute your specimen as much as you want.

Blessings
~ND

That's why I think it would be at least worth a try. But I'm EU based, so I can't test this easily.

Perhaps this thread will interest you: https://www.dmt-nexus.me...aspx?g=posts&t=21752

The first person who acomplishes this will get the Nobel Price in DMT.



Then do it uneasily

I'd try to re-engineer B. caapi, since no further extraction is needed - just chew the leaves with some orange juice.

Or better: introduce Aromatic L-amino acid decarboxylase, Amine N-methyltransferase and indole harmalatransferase (I made that up, idk how the Harmala alkaloid producing proteins are called - any idea?) into a fast growing, innocent looking plant. It's called pharming, so the idea is not completely fictional.

Addendum:

Playing with the indolethylamine N-methyltransferase model, I found out that Phenylethanolamine N-methyltransferase & Nicotinamide N-methyltransferase look very much alike, so I wonder if a mescaline/DMT producing tabacco plant is foreseeable in the near future?

Please order and reship the needed DNA in my direction.

I mean...don't you guys think that it would be worth to give it try? Doesn't seem to be THAT expensive. I may be naive or something...but what if it just like....works?

Have you seen the link regarding THC?

As a plant biologist working in a plant/biology lab I would say, of course its possible. But as already stated, it will be a lot of work and cannot be done under the radar in any kind of european research unit. Maybe it could be done, with some money, a lot of experience in the field and the spirits on your side - time will see.
What would be really nice is a DMT containing plant species that could be easily grown under different climatic conditions and easy to extract from. I believe it exists and just had to found.
Just think of Phragmites australis, which builds kind of a natural monoculture.

Phalaris grass is what you're looking for.

Blessings
~ND

This technique is utilizing site-directed mutagenesis to synthesize DMT via a genetically modified organism:

1. Introduce a gene to a host cell via a plasmid that encodes for a particular protein(s) (in this case 2 enzymes).

2. The host cell produces these enzymes from the introduced gene(s).

3. These enzymes then produce DMT from L-Tryptophan already present (or introduced) to the host cell.

4. The DMT would be extracted from the host cell using various techniques (STB, A/B, column chromatography, centrifugation, etc.)


This is a very feasible (and interesting) method of producing and extracting DMT. Although there are at least a few things to think about though before embarking on this experiment...

- The concentration DMT in the host cell after biosynthesis
- The difficulty of the extraction of DMT from the host cell
- Possibly many more...

Concentration issues:
If the concentration of DMT in the host cell is to low after biosynthesis (or lower than other sources, eg. MHRB, ACRB, Phalaris, etc.) then this method of producing DMT would not be feasible as a substitute to traditional production techniques due to cost, time efficiency, and difficulty.

Extraction issues:
It is possible that the extraction of DMT from genetically modified organisms (E. coli) could be more difficult compared to the extraction from traditional sources. Once again due to cost, time efficiency, and difficulty.


If these (and possibly many more) issues are resolved then this method of DMT production is entirely feasible. I'd love to hear how this works out for someone who is willing to give it a shot.

Yes. But I think it would be a good thing to just try this tek. Then we would SEE how much of an issue these issues really are.

I mean it sounds like everyone is saying that it's a risky, costly thing to just try on a whim, especially since those qualified are probably very busy with their careers. Hence why the suggestion that you yourself try it has been made, as you seem the most eager. No negative vibes intended btw, just hoping to clear up some confusion as to why you might be getting the responses you are. My apologies in advance for any perceived offense (none is intended, I assure you).

Absolutely no offense taken. As I said...I may be naive. That said, when you say risky and costly I don't really see those points.

Why is it risky? The procedure to introduce the DNA to the e.coli SEEMS easy.



And costly...? I'm not sure. The first custom plasmid link I clicked said "Economical: 100 µg miniprep starting from just $50". I don't know much would be needed tough.

Seems like I just miss/don't understand something, which is supposed to be difficult.



Indeed. If I just could order that plasmid, like US people can, I would already have tried it.

Well, for starters, E coli is a pretty nasty germ once ingested. So there's difinitely risk involved if you're not careful: contamination could be disastrous. Plus, having done this technique for some experiments in college, it's something takes a little practice to get right. Also $50 doesn't seem bad, though I don't know how much one would need. Hopefully someone gives this a try though, it sounds like it could be the next big thing if someone can get it to work and condense it down to a kitchen friendly recipe.

EDIT: It's one of those lab techniques that looks easy on paper but can be frustrating until you have a little practice. That being said, I'm feeling tempted now to do it. Oh lord, time will tell haha

It would certainly be an expensive project. You'd need very precise incubators, centrifuge tubes, petri-dishes, growth medium, plasmids, uL pipettes, uL pipette tips, antibiotics (which I'm not sure you can buy without a license, although I'm not sure), etc. You'd need to outfit yourself with all the trappings of a molecular or cell biology lab, and that stuff isn't cheap. One of my mentors described it all as "hideously expensive." I've never looked, maybe you could find stuff for cheaper on eBay, but there's a reason researchers compete so hard for grants and funding. You need more than pocket change to do real science.

This may be inappropriate speculation, but I could see this being of interest to some of the big drug cartels. They have the resources and brains on staff to pull this kind of thing off, and, if it were done right, they could stand to make a lot of money. Probably not with DMT, but perhaps with morphine? There was recently a big breakthrough in getting yeast to do the biosynthesis morphine - I bet there are a lot of morally dubious folks who have their eyes on that, potentially very lucrative, research (I'll let you decide if I'm talking about cartels, or pharmaceutical execs).

Blessings
~ND

This might be true for a compound as difficult to synthesize as heroin but with DMT, total synthesis is a much easier and cost effective method of obtaining mass quantities of DMT.

You are correct about your assessment of the equipment needed for the OPs experiment. You would basically need an entire molecular biology lab, which is very expensive.

More likely they would manufacture amphetamines from candida utilis. It's not breaking news, the pharmacutical industry has been doing this for years.

But mexico has very different laws regarding precurser materials, so then again --maybe they don't.

To change the subject: I've though about it, and wouldn't cloning dmt producing plant cells be a more worthwhile endeavor? procaryotic microorganisms do have that appeal. But nature already provided cells that produce substances like mescaline and dmt. Why not use those,... that's where I've been putting my efforts.

If 2 weeks culture time produced say.. 50g dmt enriched biomass then the game has already been won.

Will specialized plant cells like rhizomes or bark thrive in a LC media? I'd imagine you'd have anoxic conditions. I suppose you could just bubble O2 in like a fish tank. I don't think the plants would produce DMT though, they'd probably move towards developing another mature whole plant (every plant cell is capable of growing a whole new plant save for some weird ones in the phleom). You could probably force a tissue type with the right balance of hormones as well (I'd imagine most plant hormones are fairly soluble in water, but that is just a guess).

That's the sort of problem I was considering. Surely there is a way to achieve mitosis of an exact cell. From what I've read sofar LC could work just fine. If not then maybe culturing inside glass plates.

you have no idea...

which is all wasteful spending, and business political in nature, of the oligopoly persuasion (not unlike the corporate republic status quo).

there are a handful of major suppliers..the big fish gobbled up all the smaller fish, so to speak, and are free to charge whatever they want for their supplies. they have sole ownership over the R&D, patents, etc.


buy the equipment/supplies on ebay, get the plasmids from NEB, Promega, etc.

DIY Bacterial Gene Engineering CRISPR Kit

Kit contains enough materials for around 5 experiments or more

LB Agar
LB Strep/Kan Agar
Glass bottle for pouring plates
E. coli HME63 strain
Inoculation Loops/Plate Spreader
10-100uL variable volume adjustable pipette(1uL increments)
Box of 96 Pipette Tips
14 Petri Plates
Microcentrifuge tube rack
Nitrile Gloves
Microcentrifuge tubes
50mL Tube for measuring
Bacterial transformation buffer 25mM CalCl2, 10% PEG 8000 5% DMSO
LB Media for transformation recovery
Cas9 and tracrRNA plasmid
crRNA plasmid
Template DNA

__________

$150.00