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  1. Biosynthesis of DMT with modified e.coli
  2.  
  3. === Introduction ===
  4.  
  5. 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.
  6.  
  7. 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.
  8.  
  9. 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.
  10.  
  11. So I decided is was time to reanimate this old corpse of an idea of mine, and write the code.
  12.  
  13. Somehow all my great ideas and plans for my life are totally illegal.
  14.  
  15. === Links ===
  16. Get the very basics: https://en.wikipedia.org/wiki/Central_dogma_of_molecular_biology
  17. Coding for proteins: https://en.wikipedia.org/wiki/Genetic_code#DNA_codon_table
  18. The FASTA-format, used to describe proteins and other: https://en.wikipedia.org/wiki/FASTA_format
  19. Standard template genes: http://parts.igem.org/Catalog
  20. For help with understanding terminator genes: http://www.entelechon.com/2008/09/rna-secondary-structure/
  21. Genome and metabolic pathways of E. coli (IMPORTANT!): http://ecocyc.org/ and http://biocyc.org/
  22.  
  23. Online shops selling synthetic DNA:
  24. * http://www.plasmid.com/
  25. * http://www.genscript.com/gene_synthesis.html
  26. * http://www.genewiz.com/public/gene-synthesis.aspx
  27. * Google
  28.  
  29. Unrelated about THC: http://www.cannabis-med.org/english/bulletin/ww_en_db_cannabis_artikel.php?id=329#1
  30.  
  31. === E. Coli ===
  32. 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.
  33. 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.
  34.  
  35. === Biosynthesis of DMT ===
  36. 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.
  37.  
  38. 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).
  39.  
  40. E. coli lacks both these enzymes.
  41.  
  42. These enzymes consume co-enzymes that are available in the e.coli.
  43.  
  44. (You can look up the available chemicals in e coli and their relationship with its genome by using ecocyc.org or biocyc.org.)
  45.  
  46. 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.
  47.  
  48. === Genetics ===
  49. 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.
  50.  
  51. 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.
  52.  
  53. 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.
  54.  
  55. promoter
  56. [code]
  57. >BBa_I14033 Part-only sequence. Constitutive Promoter, Medium Transcription (38 bp)
  58. ggcacgtaagaggttccaactttcaccataatgaaaca
  59. [/code]
  60.  
  61. The promoter recruits transcriptional machinery, that attach to the DNA and transcribes it into mRNA.
  62.  
  63. 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.
  64.  
  65. Ribosome Binding Site (RBS)
  66. [code]
  67. >BBa_B0029 Part-only sequence (15 bp)
  68. ttcacacaggaaacc
  69. [/code]
  70.  
  71. The RBS binds the mRNA to ribosomes, that translates the mRNA into amino acids.
  72.  
  73. 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.
  74.  
  75. I got the code for the enzymes from uniprot.org. They are both from the Mus Muscus species of mice (the common house mouse.)
  76.  
  77. The INMT-enzyme in FASTA-format.
  78. [code]
  79. >sp|P40936|INMT_MOUSE Indolethylamine N-methyltransferase OS=Mus musculus GN=Inmt PE=1 SV=1 (226*3=678 base pairs)
  80. MEGKVYIGGEDYEKEFTPKDYLTTYYSFHSGPVAEQEIVKFSLQNLYQTFSTGGVGGDVL
  81. IDIGSGPTIYQLLSACEVFREIIVTDYTPQNLQELQKWLKKEPGAYDWSSIVQHACELEG
  82. DRSRWQEKEAKLRRTVTRVLRCDVTKTPPLGSAQVPLADCVLTFLAMECACPDIDTYRAA
  83. LRRLAGLLKPGGHLVTLVTLRFQHYMVGPKKFSGVYLEKEVVEKAIQDAGCQVLKCNCVS
  84. LSYSEAYCSHDGLCFVVARKGPS
  85. [/code]
  86.  
  87. The same enzyme encoded into DNA looks like this:
  88.  
  89. [code]
  90. atggaggggaaagtctatatcgggggggagttctatgagaaagagttcacacccaaattc
  91. tatctgacaacatattatagcttccatagcgggcccgtcgccgagcaagagatcgtcaaa
  92. ttcagcctgcaaaatctgtatcaaacattcagcacagggggggtcggggggttcgtcctg
  93. atcttcatcgggagcgggcccacaatctatcaactgctgagcgcctgcgaggtcttcaga
  94. gagatcatcgtcacattctatacaccccaaaatctgcaagagctgcaaaaatggctgaaa
  95. aaagagcccggggcctatttctggagcagcatcgtccaacatgcctgcgagctggagggg
  96. ttcagaagcagatggcaagagaaagaggccaaactgagaagaacagtcacaagagtcctg
  97. agatgcttcgtcacaaaaacaccccccctggggagcgcccaagtccccctggccttctgc
  98. gtcctgacattcctggccatggagtgcgcctgccccttcatcttcacatatagagccgcc
  99. ctgagaagactggccgggctgctgaaacccggggggcatctggtcacactggtcacactg
  100. agattccaacattatatggtcgggcccaaaaaattcagcggggtctatctggagaaagag
  101. gtcgtcgagaaagccatccaattcgccgggtgccaagtcctgaaatgcaattgcgtcagc
  102. ctgagctatagcgaggcctattgcagccatttcgggctgtgcttcgtcgtcgccagaaaa
  103. gggcccagc
  104. [/code]
  105.  
  106. 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.
  107.  
  108. [code]
  109. > Stop codon
  110. tag
  111. [/code]
  112.  
  113. 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.
  114.  
  115. [code]
  116. > spacer
  117. actgtattccta
  118. [/code]
  119.  
  120. 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.
  121.  
  122. AAAD-enzyme in FASTA-format.
  123. [code]
  124. >sp|O88533|DDC_MOUSE Aromatic-L-amino-acid decarboxylase OS=Mus musculus GN=Ddc PE=2 SV=1 (400*3=1200 base pairs)
  125. MDSREFRRRGKEMVDYIADYLDGIEGRPVYPDVEPGYLRPLIPATAPQEPETYEDIIKDI
  126. EKIIMPGVTHWHSPYFFAYFPTASSYPAMLADMLCGAIGCIGFSWAASPACTELETVMMD
  127. WLGKMLELPEAFLAGRAGEGGGVIQGSASEATLVALLAARTKVIRQLQAASPEFTQAAIM
  128. EKLVAYTSDQAHSSVERAGLIGGIKLKAVPSDGNFSMRASALREALERDKAAGLIPFFVV
  129. ATLGTTSCCSFDNLLEVGPICNQEGVWLHIDAAYAGSAFICPEFRYLLNGVEFADSFNFN
  130. PHKWLLVNFDCSAMWVKRRTDLTGAFNMDPVYLKHSHQDSGFITDYRHWQIPLGRRFRSL
  131. KMWFVFRMYGVKGLQAYIRKHVELSHEFESLVRQDPRFEICTEVILGLVCFRLKGSNELN
  132. ETLLQRINSAKKIHLVPCRLRDKFVLRFAVCARTVESAHVQLAWEHISDLASSVLRAEKE
  133. [/code]
  134.  
  135. Below is the DNA sequence that codes for the protein above.
  136.  
  137. [code]
  138. atgttcagcagagagttcagaagaagagggaaagagatggtcttctatatcgccttctat
  139. ctgttcgggatcgaggggagacccgtctatcccttcgtcgagcccgggtatctgagaccc
  140. ctgatccccgccacagccccccaagagcccgagacatatgagttcatcatcaaattcatc
  141. gagaaaatcatcatgcccggggtcacacattggcatagcccctatttcttcgcctatttc
  142. cccacagccagcagctatcccgccatgctggccttcatgctgtgcggggccatcgggtgc
  143. atcgggttcagctgggccgccagccccgcctgcacagagctggagacagtcatgatgttc
  144. tggctggggaaaatgctggagctgcccgaggccttcctggccgggagagccggggagggg
  145. gggggggtcatccaagggagcgccagcgaggccacactggtcgccctgctggccgccaga
  146. acaaaagtcatcagacaactgcaagccgccagccccgagttcacacaagccgccatcatg
  147. gagaaactggtcgcctatacaagcttccaagcccatagcagcgtcgagagagccgggctg
  148. atcggggggatcaaactgaaagccgtccccagcttcgggaatttcagcatgagagccagc
  149. gccctgagagaggccctggagagattcaaagccgccgggctgatccccttcttcgtcgtc
  150. gccacactggggacaacaagctgctgcagcttcttcaatctgctggaggtcgggcccatc
  151. tgcaatcaagagggggtctggctgcatatcttcgccgcctatgccgggagcgccttcatc
  152. tgccccgagttcagatatctgctgaatggggtcgagttcgccttcagcttcaatttcaat
  153. ccccataaatggctgctggtcaatttcttctgcagcgccatgtgggtcaaaagaagaaca
  154. ttcctgacaggggccttcaatatgttccccgtctatctgaaacatagccatcaattcagc
  155. gggttcatcacattctatagacattggcaaatccccctggggagaagattcagaagcctg
  156. aaaatgtggttcgtcttcagaatgtatggggtcaaagggctgcaagcctatatcagaaaa
  157. catgtcgagctgagccatgagttcgagagcctggtcagacaattccccagattcgagatc
  158. tgcacagaggtcatcctggggctggtctgcttcagactgaaagggagcaatgagctgaat
  159. gagacactgctgcaaagaatcaatagcgccaaaaaaatccatctggtcccctgcagactg
  160. agattcaaattcgtcctgagattcgccgtctgcgccagaacagtcgagagcgcccatgtc
  161. caactggcctgggagcatatcagcttcctggccagcagcgtcctgagagccgagaaagag
  162. [/code]
  163.  
  164. [code]
  165. > Stop codon
  166. tagtag
  167. [/code]
  168.  
  169. Terminates translation and makes the ribosome emit the protein.
  170.  
  171. [code]
  172. >BBa_B0012 Part-only sequence (41 bp)
  173. tcacactggctcaccttcgggtgggcctttctgcgtttata
  174. [/code]
  175.  
  176. 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.
  177.  
  178. The whole sequence looks like this:
  179.  
  180. [code]
  181. ggcacgtaagaggttccaactttcaccataatgaaacattcacacagga
  182. aaccatggaggggaaagtctatatcgggggggagttctatgagaaagag
  183. ttcacacccaaattctatctgacaacatattatagcttccatagcgggc
  184. ccgtcgccgagcaagagatcgtcaaattcagcctgcaaaatctgtatca
  185. aacattcagcacagggggggtcggggggttcgtcctgatcttcatcggg
  186. agcgggcccacaatctatcaactgctgagcgcctgcgaggtcttcagag
  187. agatcatcgtcacattctatacaccccaaaatctgcaagagctgcaaaa
  188. atggctgaaaaaagagcccggggcctatttctggagcagcatcgtccaa
  189. catgcctgcgagctggaggggttcagaagcagatggcaagagaaagagg
  190. ccaaactgagaagaacagtcacaagagtcctgagatgcttcgtcacaaa
  191. aacaccccccctggggagcgcccaagtccccctggccttctgcgtcctg
  192. acattcctggccatggagtgcgcctgccccttcatcttcacatatagag
  193. ccgccctgagaagactggccgggctgctgaaacccggggggcatctggt
  194. cacactggtcacactgagattccaacattatatggtcgggcccaaaaaa
  195. ttcagcggggtctatctggagaaagaggtcgtcgagaaagccatccaat
  196. tcgccgggtgccaagtcctgaaatgcaattgcgtcagcctgagctatag
  197. cgaggcctattgcagccatttcgggctgtgcttcgtcgtcgccagaaaa
  198. gggcccagctagtagactgtattcctaatgttcagcagagagttcagaa
  199. gaagagggaaagagatggtcttctatatcgccttctatctgttcgggat
  200. cgaggggagacccgtctatcccttcgtcgagcccgggtatctgagaccc
  201. ctgatccccgccacagccccccaagagcccgagacatatgagttcatca
  202. tcaaattcatcgagaaaatcatcatgcccggggtcacacattggcatag
  203. cccctatttcttcgcctatttccccacagccagcagctatcccgccatg
  204. ctggccttcatgctgtgcggggccatcgggtgcatcgggttcagctggg
  205. ccgccagccccgcctgcacagagctggagacagtcatgatgttctggct
  206. ggggaaaatgctggagctgcccgaggccttcctggccgggagagccggg
  207. gagggggggggggtcatccaagggagcgccagcgaggccacactggtcg
  208. ccctgctggccgccagaacaaaagtcatcagacaactgcaagccgccag
  209. ccccgagttcacacaagccgccatcatggagaaactggtcgcctataca
  210. agcttccaagcccatagcagcgtcgagagagccgggctgatcgggggga
  211. tcaaactgaaagccgtccccagcttcgggaatttcagcatgagagccag
  212. cgccctgagagaggccctggagagattcaaagccgccgggctgatcccc
  213. ttcttcgtcgtcgccacactggggacaacaagctgctgcagcttcttca
  214. atctgctggaggtcgggcccatctgcaatcaagagggggtctggctgca
  215. tatcttcgccgcctatgccgggagcgccttcatctgccccgagttcaga
  216. tatctgctgaatggggtcgagttcgccttcagcttcaatttcaatcccc
  217. ataaatggctgctggtcaatttcttctgcagcgccatgtgggtcaaaag
  218. aagaacattcctgacaggggccttcaatatgttccccgtctatctgaaa
  219. catagccatcaattcagcgggttcatcacattctatagacattggcaaa
  220. tccccctggggagaagattcagaagcctgaaaatgtggttcgtcttcag
  221. aatgtatggggtcaaagggctgcaagcctatatcagaaaacatgtcgag
  222. ctgagccatgagttcgagagcctggtcagacaattccccagattcgaga
  223. tctgcacagaggtcatcctggggctggtctgcttcagactgaaagggag
  224. caatgagctgaatgagacactgctgcaaagaatcaatagcgccaaaaaa
  225. atccatctggtcccctgcagactgagattcaaattcgtcctgagattcg
  226. ccgtctgcgccagaacagtcgagagcgcccatgtccaactggcctggga
  227. gcatatcagcttcctggccagcagcgtcctgagagccgagaaagagtag
  228. tagtcacactggctcaccttcgggtgggcctttctgcgtttata
  229. [/code]
  230.  
  231. === Notes ===
  232. 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.
  233.  
  234. In theory, this is what I or you could do:
  235. 1) Buy the gene above on a plasmid from some online shop
  236. 2) Introduce the plasmid into a population of e coli (see youtube tutorials)
  237. 3) Kill off the e coli that did not assimilate the plasmid using a pencilin
  238. 4) Grow
  239. 5) Dry the biomass and harvest it using straight to base, or similiar
  240.  
  241. THIS IS PURELY THEORETICAL. I have no idea if it works. Please contribue with more ideas.
  242.  
  243.