This article is a stub. As such, it may contain incomplete or wrong information. You can help by expanding it.

Disclaimer: This guide is provided for informational and educational purposes only. We do not encourage you to break the law and cannot claim any responsibility for your actions.

The use of Genetic engineering to create the Biosynthesis of DMT in modified organisms

Introduction

As of June 2017 nobody has ever made a modification in a organism to produce DMT.

This article contemplate the research that is going on in this subject.

Simplified steps to achieve the goal

Learn about the genes involved Biosynthesis of DMT in a specific organism that produce it like humans or Mimosa hostilis. Choose a organism that do not create DMT naturally like E.Coli or a specific plant. Write the plasmid digitally Send the plasmid info (digital) to any place that sell plasmid and Buy the sequence that you want Introduce the plasmid into the organisms (at home or pay a laboratoy) Grow organisms that are producing DMT Harvest dmt using some Tek

The theory - Research on how to do the gene modification

Biosynthesis of DMT in humans

Dimethyltryptamine is an indole alkaloid derived from the shikimate pathway.

the biosynthesis of DMT is relatively simple.

N,N-dimethyltryptamine Biosynthetic pathway for-dimethyltryptamine

L-tryptophan that we cosume is transformed in DMT in 4 step listed below.

Humans cannot produce L-tryptophan, so we need to eat it. Tryptophan is a routine constituent of most protein-based foods or dietary proteins. It is particularly plentiful in milk, yogurt, cottage cheese, red meat, eggs, etc.

It begins with its decarboxylation (a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2)) of L-tryptophan by an aromatic amino acid decarboxylase (AADC) enzyme

Aromatic L-amino acid decarboxylase (AADC)

(step 1). The resulting decarboxylated tryptophan analog is tryptamine.

tryptamine

Tryptamine then undergoes a transmethylation

(step 2): the enzyme indolethylamine-N-methyltransferase (INMT) catalyzes the transfer of a methyl group from cofactor S-adenosyl-methionine (SAM), via nucleophilic attack, to tryptamine.

Amine N-methyltransferase (INMT)

This reaction transforms SAM into S-adenosylhomocysteine (SAH), and gives the intermediate product N-methyltryptamine (NMT).

N-Methyltryptamine (NMT) has been found in the bark, shoots and leaves of several plant genera, including Virola, Acacia, Mimosa

[1][2] NMT is in turn transmethylated by the same process

(step 3) to form the end product N,N-dimethyltryptamine. Tryptamine transmethylation is regulated by two products of the reaction: SAH,[3][4][5] and DMT[3][5] were shown ex vivo to be among the most potent inhibitors of rabbit INMT activity.

This transmethylation mechanism has been repeatedly and consistently proven by radiolabeling of SAM methyl group with carbon-14 (14C-CH 3 )SAM).[1][3][5][6][7]

DMT and the human DNA

Dmt in humans is created with genes located in Chromosome 7.

the genes are listed below

Amine N-methyltransferase (INMT)

EntrezGene = 21743 UniProt = P40936

Template:Enzyme

In enzymology, an Amine N-methyltransferase (Template:EC number) is an enzyme that is ubiquitously present in non-neural tissues and that catalyzes the N-methylation of tryptamine and structurally related compounds.[8]

The chemical reaction taking place is:

S-adenosyl-L-methionine + an amine <math>\rightleftharpoons</math> S-adenosyl-L-homocysteine + a methylated amine

Thus, the two substrates of this enzyme are S-adenosyl methionine and amine, whereas its two products are S-adenosylhomocysteine and methylated amine. In the case of tryptamine and serotonin these then become the dimethylated indolethylamines dimethyltryptamine (DMT) and bufotenine.[9]

This enzyme belongs to the family of transferases, specifically those transferring one-carbon group methyltransferases. The systematic name of this enzyme class is S-adenosyl-L-methionine:amine N-methyltransferase. Other names in common use include nicotine N-methyltransferase, tryptamine N-methyltransferase, indolethylamine N-methyltransferase, and arylamine N-methyltransferase. This enzyme participates in tryptophan metabolism.

A wide range of primary, secondary and tertiary amines can act as acceptors, including tryptamine, aniline, nicotine and a variety of drugs and other xenobiotics.[8]



N-methylation of endogenous and xenobiotic compounds is a major method by which they are degraded. This gene encodes an enzyme that N-methylates indoles such as tryptamine. Alternative splicing results in multiple transcript variants. Read-through transcription also exists between this gene and the downstream FAM188B (family with sequence similarity 188, member B) gene. [provided by RefSeq, Nov 2010]

Enzyme encoded into DNA

atggaggggaaagtctatatcgggggggagttctatgagaaagagttcacacccaaattc tatctgacaacatattatagcttccatagcgggcccgtcgccgagcaagagatcgtcaaa ttcagcctgcaaaatctgtatcaaacattcagcacagggggggtcggggggttcgtcctg atcttcatcgggagcgggcccacaatctatcaactgctgagcgcctgcgaggtcttcaga gagatcatcgtcacattctatacaccccaaaatctgcaagagctgcaaaaatggctgaaa aaagagcccggggcctatttctggagcagcatcgtccaacatgcctgcgagctggagggg ttcagaagcagatggcaagagaaagaggccaaactgagaagaacagtcacaagagtcctg agatgcttcgtcacaaaaacaccccccctggggagcgcccaagtccccctggccttctgc gtcctgacattcctggccatggagtgcgcctgccccttcatcttcacatatagagccgcc ctgagaagactggccgggctgctgaaacccggggggcatctggtcacactggtcacactg agattccaacattatatggtcgggcccaaaaaattcagcggggtctatctggagaaagag gtcgtcgagaaagccatccaattcgccgggtgccaagtcctgaaatgcaattgcgtcagc ctgagctatagcgaggcctattgcagccatttcgggctgtgcttcgtcgtcgccagaaaa gggcccagc

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.

Aromatic L-amino acid decarboxylase (AADC or AAAD)

http://www.uniprot.org/uniprot/O88533

Template:Enzyme Template:Infobox protein Aromatic L-amino acid decarboxylase (AADC or AAAD), also known as DOPA decarboxylase, tryptophan decarboxylase, and 5-hydroxytryptophan decarboxylase, is a lyase enzyme (Template:EC number).

Reactions

AADC catalyzes several different decarboxylation reactions:[10]

L-DOPA to dopamine - a neurotransmitter

5-HTP to serotonin (5-HT) - a neurotransmitter

L-histidine to histamine - a neurotransmitter

phenylalanine to phenethylamine - a trace amine neuromodulator

L-tyrosine to tyramine - a trace amine neuromodulator

tryptophan to tryptamine - a trace amine neuromodulator

The enzyme uses pyridoxal phosphate, the active form of vitamin B 6 , as a cofactor. Template:Phenylalanine biosynthesis

Human serotonin biosynthesis pathway

Template:Clear left

Below is the DNA sequence that codes for the protein above

atgttcagcagagagttcagaagaagagggaaagagatggtcttctatatcgccttctat ctgttcgggatcgaggggagacccgtctatcccttcgtcgagcccgggtatctgagaccc ctgatccccgccacagccccccaagagcccgagacatatgagttcatcatcaaattcatc gagaaaatcatcatgcccggggtcacacattggcatagcccctatttcttcgcctatttc cccacagccagcagctatcccgccatgctggccttcatgctgtgcggggccatcgggtgc atcgggttcagctgggccgccagccccgcctgcacagagctggagacagtcatgatgttc tggctggggaaaatgctggagctgcccgaggccttcctggccgggagagccggggagggg gggggggtcatccaagggagcgccagcgaggccacactggtcgccctgctggccgccaga acaaaagtcatcagacaactgcaagccgccagccccgagttcacacaagccgccatcatg gagaaactggtcgcctatacaagcttccaagcccatagcagcgtcgagagagccgggctg atcggggggatcaaactgaaagccgtccccagcttcgggaatttcagcatgagagccagc gccctgagagaggccctggagagattcaaagccgccgggctgatccccttcttcgtcgtc gccacactggggacaacaagctgctgcagcttcttcaatctgctggaggtcgggcccatc tgcaatcaagagggggtctggctgcatatcttcgccgcctatgccgggagcgccttcatc tgccccgagttcagatatctgctgaatggggtcgagttcgccttcagcttcaatttcaat ccccataaatggctgctggtcaatttcttctgcagcgccatgtgggtcaaaagaagaaca ttcctgacaggggccttcaatatgttccccgtctatctgaaacatagccatcaattcagc gggttcatcacattctatagacattggcaaatccccctggggagaagattcagaagcctg aaaatgtggttcgtcttcagaatgtatggggtcaaagggctgcaagcctatatcagaaaa catgtcgagctgagccatgagttcgagagcctggtcagacaattccccagattcgagatc tgcacagaggtcatcctggggctggtctgcttcagactgaaagggagcaatgagctgaat gagacactgctgcaaagaatcaatagcgccaaaaaaatccatctggtcccctgcagactg agattcaaattcgtcctgagattcgccgtctgcgccagaacagtcgagagcgcccatgtc caactggcctgggagcatatcagcttcctggccagcagcgtcctgagagccgagaaagag

Construct a digital plasmid

Genome and metabolic pathways of E. coli (IMPORTANT!): http://ecocyc.org/ and http://biocyc.org/

plasmid

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/wiki/N,N-Dimethyltryptamine#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.



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.

BBa_I14033

http://parts.igem.org/Part:BBa_I14033

http://beta.labgeni.us/registries/parts_registry/?part=BBa_I14033

promoter

>BBa_I14033 Part-only sequence. Constitutive Promoter, Medium Transcription (38 bp)

ggcacgtaagaggttccaactttcaccataatgaaaca

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.

BBa_B0029

http://parts.igem.org/Part:BBa_B0029

Ribosome Binding Site (RBS)

>BBa_B0029 Part-only sequence (15 bp)

ttcacacaggaaacc



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.

spacer 1

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.

> spacer

actgtattccta

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.

Stop codon tagtag

BBa_B0012

Terminates translation and makes the ribosome emit the protein.

>BBa_B0012 Part-only sequence (41 bp)

tcacactggctcaccttcgggtgggcctttctgcgtttata



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.

Final

The whole sequence looks like this:

ggcacgtaagaggttccaactttcaccataatgaaacattcacacagga aaccatggaggggaaagtctatatcgggggggagttctatgagaaagag ttcacacccaaattctatctgacaacatattatagcttccatagcgggc ccgtcgccgagcaagagatcgtcaaattcagcctgcaaaatctgtatca aacattcagcacagggggggtcggggggttcgtcctgatcttcatcggg agcgggcccacaatctatcaactgctgagcgcctgcgaggtcttcagag agatcatcgtcacattctatacaccccaaaatctgcaagagctgcaaaa atggctgaaaaaagagcccggggcctatttctggagcagcatcgtccaa catgcctgcgagctggaggggttcagaagcagatggcaagagaaagagg ccaaactgagaagaacagtcacaagagtcctgagatgcttcgtcacaaa aacaccccccctggggagcgcccaagtccccctggccttctgcgtcctg acattcctggccatggagtgcgcctgccccttcatcttcacatatagag ccgccctgagaagactggccgggctgctgaaacccggggggcatctggt cacactggtcacactgagattccaacattatatggtcgggcccaaaaaa ttcagcggggtctatctggagaaagaggtcgtcgagaaagccatccaat tcgccgggtgccaagtcctgaaatgcaattgcgtcagcctgagctatag cgaggcctattgcagccatttcgggctgtgcttcgtcgtcgccagaaaa gggcccagctagtagactgtattcctaatgttcagcagagagttcagaa gaagagggaaagagatggtcttctatatcgccttctatctgttcgggat cgaggggagacccgtctatcccttcgtcgagcccgggtatctgagaccc ctgatccccgccacagccccccaagagcccgagacatatgagttcatca tcaaattcatcgagaaaatcatcatgcccggggtcacacattggcatag cccctatttcttcgcctatttccccacagccagcagctatcccgccatg ctggccttcatgctgtgcggggccatcgggtgcatcgggttcagctggg ccgccagccccgcctgcacagagctggagacagtcatgatgttctggct ggggaaaatgctggagctgcccgaggccttcctggccgggagagccggg gagggggggggggtcatccaagggagcgccagcgaggccacactggtcg ccctgctggccgccagaacaaaagtcatcagacaactgcaagccgccag ccccgagttcacacaagccgccatcatggagaaactggtcgcctataca agcttccaagcccatagcagcgtcgagagagccgggctgatcgggggga tcaaactgaaagccgtccccagcttcgggaatttcagcatgagagccag cgccctgagagaggccctggagagattcaaagccgccgggctgatcccc ttcttcgtcgtcgccacactggggacaacaagctgctgcagcttcttca atctgctggaggtcgggcccatctgcaatcaagagggggtctggctgca tatcttcgccgcctatgccgggagcgccttcatctgccccgagttcaga tatctgctgaatggggtcgagttcgccttcagcttcaatttcaatcccc ataaatggctgctggtcaatttcttctgcagcgccatgtgggtcaaaag aagaacattcctgacaggggccttcaatatgttccccgtctatctgaaa catagccatcaattcagcgggttcatcacattctatagacattggcaaa tccccctggggagaagattcagaagcctgaaaatgtggttcgtcttcag aatgtatggggtcaaagggctgcaagcctatatcagaaaacatgtcgag ctgagccatgagttcgagagcctggtcagacaattccccagattcgaga tctgcacagaggtcatcctggggctggtctgcttcagactgaaagggag caatgagctgaatgagacactgctgcaaagaatcaatagcgccaaaaaa atccatctggtcccctgcagactgagattcaaattcgtcctgagattcg ccgtctgcgccagaacagtcgagagcgcccatgtccaactggcctggga gcatatcagcttcctggccagcagcgtcctgagagccgagaaagagtag tagtcacactggctcaccttcgggtgggcctttctgcgtttata

Appling the theory to actually modify a organism

Buy the gene on a plasmid from some online shop

with the knowledge of the genes needed to produce DMT you have to send it to a Online shops selling synthetic DNA and buy it on a plasmid.



Online shops selling synthetic DNA:

Plasmids and Recombinant DNA Technology https://www.youtube.com/watch?v=2YxTcBimxlw

Introduce the plasmid into a population of chosen organisms

with the plasmid in hands you have to introduce it on a organism like E.coli

Grow organisms assimilate the plasmid

kill off organisms that did not assimilate the plasmid using a pencilin

Dry the biomass and harvest it using straight to base, or similiar

using some of Tek Extractions

More information

Foruns discussions

There has been a lot of reseach on online forums on how to create a organism that produces dmt using genetic engineering. Here is a list of most important threads on this subject.

DMT-Nexus

DMT-Nexus - 7/13/2008 what is the deal with genetic engineering? https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=1758

DMT-Nexus - 12/10/2008 DIY Genetic Engineering & DMT production https://www.dmt-nexus.me/forum/default.aspx?g=posts&m=63238

DMT-Nexus - 6/3/2008 a note on genetic engineering https://www.dmt-nexus.me/forum/default.aspx?g=posts&m=12907

DMT-Nexus - 12/10/2008 - DMT and genetic engeneering https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=2892

DMT-Nexus - 5/18/2011 - Genetically engineered DMT : https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=21752

DMT-Nexus - 8/10/2013 - Biosynthesis of DMT by yeast? https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=47315

DMT-Nexus - 7/22/2015 - Biosynthesis of DMT with modified e.coli : https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=66341

DMT-Nexus - 12/14/2015 - GMO/Synthehuasca: Would You Take It? https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=68577

DMT-Nexus - 4/5/2016 - Gene Modification for entheogen sustainability. https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=70324

DMT-Nexus - 5/19/2011 - Biosynthesis of Novel Tryptamines Aspirations. https://www.dmt-nexus.me/forum/default.aspx?g=posts&t=21776

reddit

reddit 2014 - Genetic Engineering and DMT production https://www.reddit.com/r/genetics/comments/1dhyk8/genetic_engineering_and_dmt_production/

reddit 2016 - Biosynthesis of DMT with modified e.coli https://www.reddit.com/r/DMT/comments/3e36ck/biosynthesis_of_dmt_with_modified_ecoli/

shroomery

shroomery - 05/26/05 - HOW TO MAKE ANY ORGANISM MAKE DMT/PSILOS https://www.shroomery.org/forums/showflat.php/Number/4227329#4227329

shroomery - 12/27/12 - Genetically engineering psychedelic plants? https://www.shroomery.org/forums/showflat.php/Number/17455243#17455243

shroomery - 02/21/06 - Psychedelics produced from GM yeasts - what's your opinion? https://www.shroomery.org/forums/showflat.php/Number/5323478

Related projects

Harvard scientists to make LSD factory from microbes : https://www.theguardian.com/science/blog/2011/jun/21/scientists-make-lsd-from-microbes

Production of THC by genetically modified bacteria : http://www.cannabis-med.org/english/bulletin/ww_en_db_cannabis_artikel.php?id=329#1

Nature.com Engineered yeast paves way for home-brew heroin : http://www.nature.com/news/engineered-yeast-paves-way-for-home-brew-heroin-1.17566

1.0 1.1 Axelrod J. (August 1961). "Enzymatic formation of psychotomimetic metabolites from normally occurring compounds". Science. 134 (3475): 343. doi:10.1126/science.134.3475.343. PMID 13685339.

3.0 3.1 3.2 Barker S.A.; Monti J.A.; Christian S.T. (1981). "N, N-dimethyltryptamine: an endogenous hallucinogen". International Review of Neurobiology. International Review of Neurobiology. 22: 83–110. doi:10.1016/S0074-7742(08)60291-3. ISBN 978-0-12-366822-6. PMID 6792104.

↑ Lin R.L.; Narasimhachari N.; Himwich H.E. (September 1973). "Inhibition of indolethylamine-N-methyltransferase by S-adenosylhomocysteine". Biochemical and Biophysical Research Communications. 54 (2): 751–9. doi:10.1016/0006-291X(73)91487-3. PMID 4756800.

↑ Mandel L.R.; Prasad R.; Lopez-Ramos B.; Walker R.W. (January 1977). "The biosynthesis of dimethyltryptamine in vivo". Research Communications in Chemical Pathology and Pharmacology. 16 (1): 47–58. PMID 14361.