Hey, sorry for the late response. Health+Family problems...

Thinking about the INMT- what about a synthetic INMT? mRNA->cDNA->cloning->synthetic INMT? Would resolve problem of eukaryotic mRNA processing?

n00b.. INMT cDNA assay kits already exist from a well known supplier.
simple Ehrlich's test of the end product would confirm presence (or lack of) of DMT.

there also exists papers (Mack & Slaytor)which describe isolation and expression of the protein from p.tuberosa

oh yea...and LC-MS/MS > GC/MS.
I've seen run times typically < 20 mins.

Eykaryotic mRNA processing? synthetic INMT You are really are really a n00b, aren't you?

INMT is your only bet to further process what TDC produces, and unfortunately the only INMT you can get (or clone if you may) is the one(s) that are already annotated.

I like when you say mRNA->cDNA etc etc, but you gotta be more precise lest not this end up in mental masturbation. I wonder where you're gonna find a tissue to clone INMT from. Human lungs? not really the most available thing around. Murine/rat tissues are far more available but still, a pain if you don't directly do animal research.

I had the mental wank session with this idea last year, clone the INMT gene into
the lacz region of e.coli. there is a paper on this somewhere on the interwebs; but I like the yeast route idea, makes better sense. e.coli does smell pretty bad.

The interesting would be not to clone it, but express it and verify it does have the wanted enzymatic activity.

Benzyme, are you aware of any published work on that deals with such questions? Also, do you have any idea where someone could realistically source (organism and tissue) the INMT gene? Do we know of any other enzymes in the universe that could do the methylation step on tryptamine?

M. hostilis as source for INMT? Should be kind of homologous to lung INMT? SWIM has a thermocycler at home, enzymes... etc. Just try, try, try? Like looking for the needle in the bale of straw?

AH, and you did not answer the synthetic gene route, except with noobing me again. Maybe i did not correctly expressed myself. I meant to order a synthetic sequence coding INMT?

ok PROFESSIONALS , ask your self a question. what is it good for to talk about something that you will never finish. lets be honest non of you will EVER make any single genetically engineered spirit molecule.

while you are wasting your time and energy on this I will make dmt in one day from a plant without any diploma its that simple

maybe you should use your intelligence on something more real. something that will solve some real problem in this world.

no offence folks

Because:

1- Who's to say this project will never be done? Based on what do you make this judgement? Do you know the qualifications of the people posting here or is this area your expertise?

2- One day relativity theory was just a theoretical idea, and years later it became somethign that is used every day by physicists, that had thousands of experiments confirming. Maybe same happens here

3- Even IF it could never be done, it's an interesting theoretical subject. We also talk about trip reports and philosophical subjects that do not have a direct connection to everyday reality, but does that mean it shouldnt be discussed? If you personally dont want to, why post here in the first place? I mean, I would understand if you would participate with questions, such as "do you think this is doable", or whatever, but you are already coming here making a judgement saying its not gonna work and posting in a sort of preachy attitude that you know better what they should be focusing their energy on. In that case better not post at all and keep to threads that interest you, no? Or speak for yourself, and post in ways you feel are constructive.

I only expressed my opinion on this matter. I still think its just waste of time. This thread looks to me like few people just showing off. Most of the people on this forum have no slightest idea what are they talking about. Well I mean when they go to details. I guess its not easy to explain those details to non professional.

By the way last replay was in July 2011 which only indicates that this subject is not interesting or really very hard to be done or done at all as was mentioned in the thread by experts.

Anyway I do apologise if I offended someone by my comment. It was not my intention. Obviously my intention was not understood but that's ok I will not post more comments in this thread and if you feel like delete it I have no problem with that.

Good luck with the project who ever will try to really do it.

you rehashed a seven month old thread just to troll it?

no I found link to this thread in new thread so I read it and then said what I though about it

But yes if you call trolling to express opinion then yes I trolled it

As I said I apologised to all who needed it

END

We are not done here Ideas still exists - but no time to realize...

You've had almost a year!!

Yeah that sucks - project is at full stop....



....Case will be opened in May

obviously, such an undertaking is not cheap. a lot of the expenses will be for purification and characterization, not to mention reagents.

money is not the problem - tryptophan is not expensive, vectors, enzymes and machines are available... at least the "proof of principle" would not cost the world.

It would. I'm with benzyme in this as money as well as time is the main problems.

If I were to put it down in a research grant or something I'd give it no less than 1 year's of full-time lab time to accomplish. Do not be fooled into thinking it'll take less just because it may feel "straightforward" or "intuitive".

Just as we speak, a supposedly "simple and straightforward" series of genetics experiments at my work has turned into a year-long nightmare... Never underestimate

I'm all however into entertaining ideas (yes, with gory details) of how things could be done, given that one has a lab bench, time and money. Any crazy mol biol takers? As already discussed, there are issues with regard to which tryptophan decarboxylase (easy to pich one) and which INMT (harder to pick one) to use.

Being in any research group with funding (of course for other research) and a lab with the usual equippment is enough.
Enzymes, vectors, hosts and reagents are always around?
Ok, time - i give you that.
How much time is required for a "proof of principle"? One month max? I'm not talking industrial size production, just the proof of principle! Once this is figured out, you have all the time to enhance

Of course only when you're lucky - like Infundibulum pointed out "simple and straightforward" means in biology "you gonna loose some hair over this".


So what we get out from ncbi and this thread ?


1. AADC
C R Acad Sci III. 1997 May;320(5):349-58. Active rat aromatic-L-amino acid decarboxylase as a fusion protein in Escherichia coli. Jebai F, Thibault J, Krieger M.

I must say, I did't read the full paper - but looks prommising.

Anyways - microorganisms have AADC, like mentioned earlier in my mental masturbations (m. loti; mlr4653)
So we can agree that the first step, decarboxylation, is not our biggest concern.


2. INMT
Rabbit Lung Indolethylamine N-Methyltransferase cDNA AND GENE CLONING AND CHARACTERIZATION Michael A. Thompson and Richard M. Weinshilboum

Well, those guys used COS1 cells as expression host - [uh, bad news, DMT seemes to inhibit INMT activity]
But benzyme mentioned some other sources, p. tuberosa...

I think it would be much more confortable using existing cDNA libraries (rat, mice, zebrafish)rather than purify mRNA from lung tissues

Good!!!

But wouldn't that be better to go for the tryptophan decarboxylase from rice seedlings, like this http://www.ncbi.nlm.nih.gov/pubmed?term=18776677 ? That's basically a good deal of the work already done and characterised. The plasmid can already be acquired by the group that made this research. I truly do not worry much about this part,

INMT is the pain. Cloning from rabbit, rat, mouse lung is the most straightforward, but as the paper you mentioned describes, INMT is inhibited by its end product, dmt. But this is the most promising way to go; first, even if you get a small amount of dmt produced then you proved your "proof of principle" and then you have a system you can tweak to perfection. Second, you have no idea about INMT from phalaris or other plants, genome resources are scarce and there's not much you can do (unless you enjoy constructing a phalaris library, fish out plant INMT homologues just to find out they do not do what you like...) The methyltransferases in plants that do this type of job are pretty much uncharacterised to hypothetical.

My first bet would therefore be on the lung INMT, unless someone has better candidates. We'll also need to tag it somehow to be able to detect it in western blots. Same goes for TDC.

Are you still