Author Topic: Mutating Organisms. How? (Read 477 times)

Vesp · « **on:** July 25, 2010, 02:34:57 AM »

There are tons of mutanogenic, carcinogenic, and teratogenic substances - I always read about them, and you can find lists of them all over the place - However I am having a bit of an issue on what one is active enough to effectively induce mutations but isn't so dangerous that it puts my life at danger due to it being something like methyl iodide, or some of those other crazy compounds.

I have a few interests in mutating organisms. Specifically dealing with fungi, plants, and flies.

My first choice would be UV light - assuming I could find a strong enough source of UV light that would allow me to cause mutations. I've looked for light bulbs but I'm not really seeing any that appear to have the purpose of inducing mutations, and I am not sure the sterilizing sort are really going to be effective.

I've read that things such as HNO2 will cause genetic mutations - I can imagine that this would work rather well when dealing with liquid cultures of mycelium, etc if it were added and incubated for a while than samples were taken from it which could than be screen for things such as increased alkaloid content, etc. hydroxylamine might also work for this purpose.
Even things like caffeine, cadmium, lead, mercury, high and low temperatures, etc I hear are supposed to cause mutations in smaller organisms but I don't know at what rate, if is practical for my purpose etc... I can't find much info on it either.

Hopefully one of you can find more info on it?
Perhaps simpler things such as toluene will induce mutations in fungi?

I'd like to learn of some other OTC or fairly easy to get/cheap chemicals - or UV source that can effectively induce mutations. It would be great if I could find some instructions of procedures that are commonly used in the laboratory on how to induce mutations in various organisms.

I'll try to add more later if I come across much, but it just doesn't seem I know the terminology to find what it is I am looking for, I guess...

Edit:
http://www.shroomery.org/forums/showflat.php/Number/475085#475085
http://www.phys.ksu.edu/gene/a3.html

I'm thinking adding a coumarin derivative to the mix + the UV ought to make it much more sensitive - I believe coumarin type compounds help induce UV mutations, right?
http://en.wikipedia.org/wiki/Giant_Hogweed#Phototoxicity
It is found in other things like parsnip, etc I believe... I'll look into it, maybe make a crude extraction if i can find a plant around here that has enough in it.

Vanadium · « **Reply #1 on:** July 25, 2010, 05:38:48 AM »

Hydroxylamine is probably your best bet unless you have access to benzene. That's how they did all their mutations back in the day.

However I must agree with some of the posters in that thread in saying that it's a shot in the dark to try to do this. There are much more effective mushroom-related ways of spending your time.

Tsathoggua · « **Reply #2 on:** July 25, 2010, 06:20:52 AM »

More effective in what sense?

We are talking of Claviceps mainly here, I think, and mutation up the wazzoo is more or less mandatory if one desires a high yielding strain.

Vesp · « **Reply #3 on:** July 25, 2010, 06:31:21 AM »

I am mostly thinking ergot - but we'll see. Also considering trying to make a wingless house fly for my pet frogs in the winter, but that is not really important

Also If I ever get to be able to do micro-propagation of plants - It again might come in useful. But ergot is mostly what I am thinking.

It seems to me that it should not be "ALL" that hard to mutate the ergot and test for various strains that have higher alkaloid content. By taking a drop of well mixed/agitated ergot that has hopefully been mutated by a compound or UV light present in it by streaking the agar plates -- multiple agar plates --- leading to hopefully tons of varying colonies of mutated ergot. Like this:
[youtube]http://www.youtube.com/watch?v=yW56Ho1nEWQ&feature=related[/youtube]

Once that is done and the colonies are of a decent size the reagent that is made of dilute FCl3 and H2SO4 in water will be sprayed on it, and than I will try to find colonies that appear a darker blue than others - implying that there is a higher alkaloid content. That strain will than be cultured and the process repeated - or streak a petri dish to isolate the strain which produced the most alkaloids, if perhaps there ended up being more than one strain in the colony.
It will be a lot of work, if it ever happens -- but It sounds fun, involves biology, chemistry and is legal but is also of questionable nature -- so it is right down my ally. I'm just trying to work everything out, and get a few things going before I start.

Tsathoggua · « **Reply #4 on:** July 25, 2010, 07:21:44 AM »

Just make sure to isolate a portion of the agar plate first and spray that, it would be a real bitch to have isolated a hiroshima-grade strain only to cover it in H2SO4 and toast the crap out of your lil babies.

embezzler · « **Reply #5 on:** July 25, 2010, 10:50:22 AM »

I would suggest 5-bromouracil if you can get your hands on it. Due to its structure it will get incorporated into the genome like a normal nucleic acid and cause mispairing on subsequent divisions and transcriptions. Maybe it can be synthesized easily enough I'm not sure. The copy and paste job I lazily performed gives a quick intro to the theorey and the attached article demonstrates it in practice. It's from the 50's so it should be of sufficiently low tech to work with limited resources.

hxxp://web.virginia.edu/Heidi/chapter29/chp29frameset.htm

Quote

Base analogs that become incorporated into DNA can induce mutations through changes in base-pairing possibilities. Two examples are 5-bromouracil (5-BU) and 2-aminopurine (2-AP). 5-Bromouracil is a thymine analog and becomes inserted into DNA at sites normally occupied by T; its 5-Br group sterically resembles thymine's 5-methyl group. However, because 5-BU frequently assumes the enol tautomeric form and pairs with G instead of A, a point mutation of the transition type may be induced (Figure 29.25). Less often, 5-BU is inserted into DNA at cytosine sites, not T sites. Then, if it base-pairs in its keto form, mimicking T, a C-G to T-A transition

Figure 29.26 × (a) 2-Aminopurine normally base-pairs with T, but (b) may also pair with cytosine through a single hydrogen bond.

ensues. The adenine analog, 2-aminopurine (recall adenine is 6-aminopurine) normally behaves like A and base-pairs with T. However, 2-AP can form a single H bond of sufficient stability with cytosine (Figure 29.26) that occasionally C replaces T in DNA replicating in the presence of 2-AP. Hypoxanthine (Figure 29.27) is an adenine analog that arises in situ in DNA through oxidative deamination of A. Hypoxanthine base-pairs with cytosine, creating an A-T to G-C transition.

Figure 29.27 × Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A-T to G-C transition.

Chemical Mutagens

Figure 29.28 × Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine. (b) Nitrosoamines, organic compounds that react to form nitrous acid, also lead to the oxidative deamination of A and C. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine. The result is a C-G to T-A transition. (d) Alkylation of G residues to give O6-methylguanine, which base-pairs with T. (e) Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards. Note that nitrosoamines are mutagenic in two ways: they can react to yield HNO2 or they can act as alkylating agents. The nitrosoguanidine, N-methyl-N'-nitro-N-nitrosoguanidine, is a very potent mutagen used in laboratories to induce mutations in experimental organisms such as Drosophila melanogaster. Ethylmethane sulfate (EMS) and dimethyl sulfate are also favorite mutagens among geneticists.

Chemical mutagens are agents that chemically modify bases so that their base-pairing characteristics are altered. For instance, nitrous acid (HNO2) causes the oxidative deamination of primary amine groups, found in adenine and cytosine. Oxidative deamination of cytosine yields uracil, which base-pairs the way T does and gives a C-G to T-A transition (Figure 29.28a). Hydroxylamine specifically causes C-G to T-A transitions because it reacts specifically with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine (Figure 29.28c). Alkylating agents are also chemical mutagens. Alkylation of reactive sites on the bases with methyl or ethyl groups alters their H-bonding and hence base pairing. For example, methylation of O6 on guanine (giving O6-methylguanine) causes this G to mispair with thymine, resulting in a G-C to A-T transition (Figure 29.28d). Alkylating agents can also induce point mutations of the transversion type. Alkylation of N7 of guanine labilizes its N-glycosidic bond, which leads to elimination of the purine ring, creating a gap in the base sequence. An enzyme, apurinic acid endonuclease, then cleaves the sugar-phosphate backbone of the DNA on the 5'-side, and the gap can be repaired by enzymatic removal of the 5'-sugar phosphate and insertion of a new nucleotide. A transversion results if a pyrimidine nucleotide is inserted in place of the purine during enzymatic repair of this gap. A number of alkylating agents are shown in Figure 29.28e.

Insertions and Deletions

The addition or removal of one or more base pairs leads to insertion or deletion mutations, respectively. Either shifts the triplet reading frame of codons, causing frameshift mutations (misincorporation of all subsequent amino acids) in the protein encoded by the gene. Such mutations can arise when flat aromatic molecules such as acridine orange (see Figure 12.16) insert themselves between successive bases in one or both strands of the double helix. This insertion or, more aptly, intercalation, doubles the distance between the bases as measured along the helix axis. This distortion of the DNA (see Figure 12.16) results in bases being inappropriately inserted or deleted when the DNA is replicated. Disruptions that arise from the insertion of a transposon within a gene also fall in this category of mutation.

(it probably sounds patronizing given the thread title but caution needed with this one, it's a lot more difficult to remove mutations)

Tsathoggua · « **Reply #6 on:** July 25, 2010, 02:06:27 PM »

Why would anybody wish to remove mutations? it is the very thing we are aiming for in this case.

Don't like the mutation? culture plate can go in the bin. There are going to be thousands potentially tens of thousands of mutations that either produce vastly less alkaloid, no alkaloid or which are lethal to the fungus, key here is willingness to work very carefully with vicious poisons, and patience in culturing, testing for production with van urk reagent or FeCl3/H2SO4 or perhaps vanillin/FeCl3/H2SO4 if 4-DMAB is unavailable. And then yet more donkey work in growing out promising looking strains to see just how much they can be made to yield.

embezzler · « **Reply #7 on:** July 25, 2010, 02:53:21 PM »

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Why would anybody wish to remove mutations? it is the very thing we are aiming for in this case.

If that mutation was in you not in the microorganism you would want to

The rest of your post sums up pretty accurately the level of work that is going to be involved in isolating a useful strain. Ideally in the isolation of microogranisms with desired metabolic paths or products a culture media would be modified to include a colour change reaction so that if the desirable product was produced then it would be visible etc. In a higher tech environment the thing to do would be to transfer the gene of interest with a fluorescent reporter or antibiotic resistance gene so that expression could be readily visible.

Sedit · « **Reply #8 on:** July 25, 2010, 03:02:21 PM »

Assuming Tsathoggua that the mutation occured in the organism and not yourselflol you typed it at the exact same time embezzler. Thats when a real problem coul present itself. Also isolation of the mutated organism could be a rough task. Even if it has a higher alkaloid content like you want how will you know? They will not exactly hold up a sign saying the mutation was successful in the direction you want it to go. It would boil down to very tedious work of sampling the agar mm by mm and reculturing each area praying that the mutation you want is the dominate speciman in that area cultured.

Random mutation always seemed like a royal bitch to me although it would be possible and has been done in the past before more accurate methods of inducing mutation arose.

What about Benzyl chloride? IIRC this was one of the first substances used to induce mutations in cultured media. Im unsure as to the mechanism by which it causes them though.

The works that embezzler included give a rough idea about the best way to go about attempting a random mutation by gathering as much as you know about the genome and seeing which basepair you want to mutate and using the appropriate substance to begin the tedious work with. It would still be random as hell but it may be more direct then other forms of random mutations.

Tsathoggua · « **Reply #9 on:** July 25, 2010, 03:34:18 PM »

Heh, I hadn't considered that it would be the experimenter being mutated. I have no wish to become a slightly past teenage mutant ninja clandestine chemist, working with that kind of reagent, arm length gloves, gas mask, and hazmat suit are coming out to play.

Apparently unlike many strains, reverted auxotrophs that yield decently are quite stable too, anyboidy know if it is possible to direct for reversion of auxotrophy? perhaps by culturing several generations of fungus to weed out the originals with badly damaged DNA thanks to the mutagenic nasties it got bathed in then gradually lowering the required nutrient in question, Eg for a methionine auxotroph high yielder, slowly lessen the amount of methionine available in the plates (or where applicable perhaps use a metabolic poison) over several generations, giving the fungus time to slowly adapt and revert back to a heterotrophic strain.

I would imagine benzyl chloride just alkylates and possibly intercalates DNA. Not the most stable of compounds either in an aqueous environment, not to mention it will hydrolyse to HCl, and somehow, I doubt Claviceps will take too kindly to being stewed in acid, I know I wouldn't.

Hehe this is just the sort of task that an autistic eye for detail and systematizing ability are going to come in really handy.

Vesp · « **Reply #10 on:** July 25, 2010, 07:05:28 PM »

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Just make sure to isolate a portion of the agar plate first and spray that, it would be a real bitch to have isolated a hiroshima-grade strain only to cover it in H2SO4 and toast the crap out of your lil babies.

That is, if it kills it - I believe there is a good chance that it will only kill the top portion, but since most myceliums have the ability to go deeper into the agar mixture than bacteria, and hopefully the solution you should be able to simply turn it over and scrape a living sample of the good strain out on the other side. - Ideally and in theory, at least! if that isn't possible, placing a petri dish on top of another and allowing it to incubate for a day ought to get the colonies growing in the same place on the other petri dish, which can than later be used to take your sample of that colony -- I doubt that it will be needed and the first method I believe was mentioned in the book you sent me, or some other file I was reading regarding ergot.

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Also isolation of the mutated organism could be a rough task. Even if it has a higher alkaloid content like you want how will you know? They will not exactly hold up a sign saying the mutation was successful in the direction you want it to go. It would boil down to very tedious work of sampling the agar mm by mm and reculturing each area praying that the mutation you want is the dominate speciman in that area cultured.

I will know it has a higher alkaloid content due to the spraying of a reagent that changes color based on the concentration of alkaloid -- more alkaloid, darker blue. So just take the colony that is the darkest blue and re culture making sure it is a single strain, than attempt to mutate it again and compare for darker blue ones, or ones that seem to grow more favorably.

If you watched the embeded youtube video you'll get what I am saying more so but it seems to me that it would not be hard to take say 1ml of mutated ergot culture solution than dilute it down to say 10ml of solution, in which you than take say 20 petri dishes and perform the smears to isolate multiple colonies that all have different genetics, hopefully some with increased alkaloid production ability, which would be determined by spraying a solution of dilute H2SO4 and FeCl3 onto it.

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There are going to be thousands potentially tens of thousands of mutations that either produce vastly less alkaloid, no alkaloid or which are lethal to the fungus, key here is willingness to work very carefully with vicious poisons, and patience in culturing, testing for production with van urk reagent or FeCl3/H2SO4 or perhaps vanillin/FeCl3/H2SO4 if 4-DMAB is unavailable. And then yet more donkey work in growing out promising looking strains to see just how much they can be made to yield.

Yes, but if I am lucky it will be like most myc and a master culture can be made which would be able to last a year or close to a year without the need to re-culture it.
This is going to be a long term project obviously and I assume I can work on it intermittently if the need arises, but I think I'd work on it till I got what I was wanting.

There will also be thousands and thousands of mutations that do nothing to the ergot fungus at all - if ergot genetics are anything like humans or most other organisms.. most of the DNA with in us are not even translated into proteins or are used at all - so a lot of the mutations that will be induced will not even be read and will not effect the organism - a bonus is also the fact that you might mutate it into reading a lot of its un-used genes, which would lead to much more complicated outcomes than you'd expect with a simple mutation. I believe...

embezzler, thanks for that info! Helped me a lot. Seems that maybe the nitrous acid could be a dilute solution, and shaken with the ergot culture medium - eventually neutralized (Do nitrite salts also cause mutations?)- assuming it wasn't killed by the pH, it would have a lot of the base pairs messed up.

I know one thing is for sure though - I'd have a lot more confidence if I could find a cheap supply of polypropylene petri dishes - they're plastic so they should he cheap and since they are polypropylene they are autoclave-able/pressure cooker-able. I can't find them, for any decent price, and few places offer them.. Only the cheap one time use polystryrene or the break the bank pyrex glass ones.

Edit: Rumor has it that Lysol induces mutations - this is the active ingredient in Lysol. http://en.wikipedia.org/wiki/Benzalkonium_chloride
Not to sure if it is all that mutagenic - could just be toxic.

Tsathoggua · « **Reply #11 on:** July 25, 2010, 09:55:32 PM »

Heh, it would be a right son of a bitch to have to buy enough pricy borosillicate glass petri dishes when one intends to be running a thousand plus plates side by side, I know the feeling, usually being poor as shit and all that.

I'l give it a shot with hydroxylamine though at some point, I need some anyway for oximation of some xxxxx for my recent brainwave/brainfart (depending on how the results are) involving a legal (in the UK) N-tBOC protected MDxx prodrug.

Vesp · « **Reply #12 on:** July 26, 2010, 05:07:17 AM »

If I had those plastic reusable ones, I'd only need to do it like 100 at a time in batches fill I found what I was looking for... Not to expensive..

embezzler · « **Reply #13 on:** July 28, 2010, 08:07:06 PM »

Well vesp, I would seriously be considering a genetic engineering approach to this mutation since random mutations dont ever have to yield the alkaloids you want. You are relying on probability but it could take years. If you are determined then adding in the sequence of interest is the way to go.

Has the genetic sequence been released for the target gene? Random mutations were the only option at the time so I think that you should embrace the here and now.

It's probably too complicated a pathway to transfer to bacterial cells but there may be yeasts that can be modified to express the gene of interest and it may just be possible to do that by OTC means with restriction enzymes isolated from environmental or non-pathogenic (and hence easily obtainable) microorganisms.

If the sequence is available publically (and I would be surprised if it wasn't) I would suggest doing a blast search and see what other organism it turns up in and with restriction enzymes it could be cut right out and put in a suitable vector for transfection.

It's probably too complicated a pathway to transfer to bacterial cells but there may be yeasts that can be modified to express the gene of interest and it may just be possible to do that by OTC means with restriction enzymes isolated from environmental or non-pathogenic and hence easily obtainable microorganisms.

hxxp://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome

One other technology that will be of interest to budding microbiologists out there that I haven't seen discussed and without violating the no sources rule I'll describe them as best I can. There are identification plates available which are plates containing approximately 20 wells that each contain a different reagent. The intention is that an aliquot of culture medium containing the microogranism of interest is pippetted in and allowed to incubate overnight. A profile of the fermentation and metabolic colour changes is then generated from each well and this is used to provide a very highly specific identification for the microorganism based on its metabolome. They dont come too cheap but if one were to purchase one I'm pretty sure that the reagent list could be copied and future ones could be home made. These are widely used by lazy clinical microbiologists if you know any.

I have linked to the holy grail of molecular biology documents. get it while its hot. This will detail how to do just about everything with nucleic acids or proteins. Current protocols in molecular biology, 4,000 pages of greatness.

http://www.uploadcity.com/?f=8881818&t=Current_Protocols_in_Molecular_Biology.pdf&

http://rapidshare.com/files/163403830/Current_Protocols_in_Molecular_Biology.pdf

Need Password? Check the source of the link:
http://www.biotechnolog.pl/forum/temat-2231.htm

P.S. I know you know this but those spot tests are also non-specific so a more intense colour doesn't necessarily mean that you have more of what you are looking for. They are a useful field test and they will tell you that you DONT have what you seek but they are not a viable method of telling what you do have. that you DONT have what you seek but they are not a viable method of telling what you do have.

Vesp · « **Reply #14 on:** July 28, 2010, 10:10:02 PM »

Thank you very much for the input, and especially the book. I'll be sure to look into that much more - it sounds like it is just what I've been looking for.

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Well vesp, I would seriously be considering a genetic engineering approach to this mutation since random mutations dont ever have to yield the alkaloids you want. You are relying on probability but it could take years. If you are determined then adding in the sequence of interest is the way to go.

I will consider it, but for me I think mutations is about the only option, and it will at least give me some good practice in biology lab type stuff. However, the input is very helpful and I will continue to research that out and look at the book you've linked too -- it might be more in reach than I believe - however, for someone that has done very little sterile work, very little culturing and is a noob to biology in general, it does seem to be a bit out there for me at least.

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P.S. I know you know this but those spot tests are also non-specific so a more intense colour doesn't necessarily mean that you have more of what you are looking for. They are a useful field test and they will tell you that you DONT have what you seek but they are not a viable method of telling what you do have. that you DONT have what you seek but they are not a viable method of telling what you do have.

What you are saying is that the tell that alkaloids are present, but not what type?

I honestly do not care what the alkaloids that the ergot produced are -- that is, I don't care if they can be used for a certain synthesis or not. I just like the idea of changing an ergot strain into another one that does produce higher alkaloid content -- and in a post around here somehow there is a pdf file about the FeCl3, H2SO4 solution and it claims that the darker the blue the higher the alkaloid content is... That I believe is all that is really needed since you can alter the alkaloids produced by how much oxygen they get - less oxygen, you end up with alkaloids that are less oxidized, etc. Either way, it doesn't matter to me -- it isn't like I ANY intent on even attempting to turn the alkaloids produced via ergot into LSD or a migraine headache medication, let alone ever doing it. I just want to play around with it, learn a bit, and perhaps inspire others to do the same... and maybe they can do something else with it.

Also i don't think it would be that difficult to screen for thousands of mutations.
perhaps I am looking at this the wrong way.. but I imagine that if a mutagen is added to a liquid culture of ergot (LC) and allowed to incubate a bit, than a tiny bit of the mutants were smeared on a hundred or so re-usable petri dishes - hopefully giving many different strains on the ergot in each petri dish, you could find a few that give higher alkaloid content, especially since you could use the few hundred petri dishes several times. say 100 (petri dishes) x 5 (re-using the 100 dishes 5 times) x 10 (different colonies per petri dish) = 5000 different strains.
5000 different strains isn't that much at all considering how many genes there are -- however, each strain would likely contain many different mutations (I'd make sure of it

), but considering how many genes must play a roll in making and regulating the alkaloid production and content it seems like the alkaloid production could be increased via several ways, not just increased expression of a single gene - though again, I could be wrong. Think about how many things can affect the height of a human however - if the goal is to make a small human, it may not be only effecting the production of growth hormone, but also the protein which it actives, the ease of calcium uptake (i.e can't grow with limited bone growth) and many other factors I bet.

Edit: Sweet! I found a perfect fix for cheap petri dishes, or so I believe... they sell 8 oz deli containers for 250 w/lids for about 30 dollars - 12 cents a piece. They are clear and made of polypropylene, they're very wide (noticably wider than the 100mm petri dishes) and are plenty deep -- polypropylene can handle pressure cooking, so they are re-useable. The agar can boil in them so you know it will be sterile. The only problem is that they are air tight and so they collapse upon cooling - however this ought to be easily fixed by a small hole in the lid or perhaps a larger ...say 2x2cm hole w/tyvek paper glued around it using RTV silicone glue.

250 of those could be used at least 5 times with out much problems and if something arose where you needed more you could get a few at the local deli for free...

consider 250 containers with 10 unique colonies in them used 5 times. 12500 different mutants (hopefully!) -- perhaps that is being too generous? How hard is it to get different strains of fungi within one petri dish?
That is a lot of nutrient mix also.. say it uses .5 oz per plate -- that is nearly 10 gallons of PDA 20 grams of agar per liter of mix -- 1.5 pounds of agar. However 2lbs of agar is only 40 dollars. OK -- so that is getting expensive for my budget, since the sugar, and other chemicals will be needed as well. That alone will use up at least a few hundred dollars... --but that is if you really need to do 1250 petri dishes... which might to wayy to many or wayy to few...

Vesp · « **Reply #15 on:** July 29, 2010, 09:16:36 AM »

From a PM I got:

Quote

I have some statistics somewhere, let me go and dig out my hidden binder full of papers (never do keep it where the house might get searched)

Righty ho, lets see what we have...

Highly efficient mutation of Claviceps purpurea using protoplasts, applied and environmental microbiology sept 1983 p 580-584 (vol 46 no.3)

'Claviceps purpurea ATCC 20102, which is aconidial under laboratory conditions was grown in submerged culture in the presence of mutagens and various nutritional additives. Protoplasts from strain ATCC 20102 were prepared and regenerated on solid medium to obtain colonies from single cell units. Frequencies of auxotrophs and high yielding mutants was on the order of 1-2%. Some of the auxotrophic mutants derived from strain ATCC 20102 were constantly segregating prototrophs. High alkaloid producing derivatives showed sclerotia like morphology and violet-brown pigmentation, in contrast to the parent strain, some of them showed segregation sectors when grown as giant colonies.

Mutagenesis of strain 1029, isolated during this study and having an increased level of alkaloid synthesis and sclerotia-like cell morphology were all stable with respect to their genotypes. However a large proportion of colonies derived from regenerated protoplasts, even in mutagen free controls showed a lowered level of alkaloid production and were morphologically similar to the wild type.

The wild type they used yielded around 15mg of ergotamine+ergokryptine per liter of medium, which is pretty fucking shit if you ask me, after 7-10 days of fermentation (not immobilised in alginate), but from strain 1029 they got 450-700mg/liter in the same time which ain't half bad, although personally I am not going to be content with less than a gram end yield of lysergic acid/isolysergic acid per liter of medium, 700mg aint shit if I'm going into the RC business.

Mutagen conc (nitrosoguanidinium) 0.067mM produced 2 high yielding colonies out of 64

0.135mM NTG-78 colonies gave 1 high yielding mutant
repeat of 0.135mM NTG and 76 colonies gave 1 high yielding mutant
0.271mM NTG-79 colonies gave fuck all
0.067mM NTG-90-1 high yielding mutant
26.5mM ethyl methansulfonate gave 1 in 96 colonies
16.1mM EMS gave 1 in 85
26.5mM EMS gave 1 in 92

Frequency of production of auxotrophs in ATCC 20102:

NTG 0.135mM: 3 Cys/Met, Met or Arg auxotrophs in 200 colonies tested
NTG 0.202mM: 3 Lys, Met, Ile auxotrophs in 80 colonies tested
NTG 0.217mM: 5 Cys/Met, Met, adenine, nicotinic acid auxotrophs in 517 colonies tested
EMS 16.1mM: 2 Cys/Met, Met auxotrophs in 100 colonies tested
EMS 26.5mM: 21 Cys/Met, Met, aromatic amino acids, 4-aminobenzoate, nicotinic acid, Leu, adenine, His+adenine auxotrophs in 1040 colonies tested

Influence of mutagens on induction of mutations affecting alkaloid synthesis of strain 1029:

Alkaloid-b
(mg L -1) no.mutants in experiment:
1 2 3 4 5 6
<5 30 23 37 26 32 21
5-75 21 16 14 25 32 14
75-150 18 7 3 5 3 2
150-225 10 2 9 8 8 7
225-300 4 2 6 7 3 6
300-400c 12 10 12 11 9 9
400-500 4 1 1 2 2 4
500-600 0 0 0 1 1 1
600-700 0 0 0 1 1 1

experiment 1- 10mM EMS in innoculum medium
experiment 2- 0.135 NTG in innoculum medium
experiment 3- no mutagen in innoculum medium
experiment 4- 10mM EMS in T-25 medium
experiment 5- 0.135mM NTG in T-25 medium
experiment 6- no mutagen in T-25 medium

b-measured after 8 daus pf growth in innoculum medium
c-level of parent strain grown under the same conditions (innoculation with piece of mycelial mat)

That helpful?

So -- it seems like it isn't that hard to get a strain that produces 700mg per liter. Right? Its pretty late when I read this...

Tsathoggua · « **Reply #16 on:** July 29, 2010, 10:17:29 AM »

From the stuff I sent you, looks that way.

I'm not happy stopping there, I KNOW big pharma has strains that crank out upto 8g/liter

That is the sort of performance I am after, I know a lot has to do with optimizing the medium, coupled with immobilizing the cells in alginate, adding fluoroinert type oxygen carrier emulsions, adding tweens, etc.

Think once high yield strains are isolated, then selective crossbreeding, coupled with further mutations on the progeny might help get there?

Vesp · « **Reply #17 on:** July 29, 2010, 05:48:27 PM »

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Think once high yield strains are isolated, then selective crossbreeding, coupled with further mutations on the progeny might help get there?

Totally -- keep in mind also that the medium on which they got the 700mg per liter strain was much less than ideal as you pointed out. Some of the things such as the addition of biotin and arsenate salts nearly double the alkaloid production. 1.4g/l is getting up there isn't it?

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Mutagen conc (nitrosoguanidinium) 0.067mM produced 2 high yielding colonies out of 64

This shouldn't be to hard to accomplish if this ratio says anything... especially if you do it many times to get multiple high yeilding colonies...

Does anyone have any more info on nitrosoguanidinium? I can't find much on it. I am assuming it is a salt of sorts of nitrosoguanidine, which is perhaps made from the addition of nitrous acid to nitroguanidine. Nitroguanidine is easily made from sulphamic acid and urea as posted on SM by Axt.

embezzler · « **Reply #18 on:** July 29, 2010, 06:54:08 PM »

@tsathoggua

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I'm not happy stopping there, I KNOW big pharma has strains that crank out upto 8g/liter

If they do it is most likely an engineered organism produced exclusively for its metabolic pathways.BTW do you have a ref for the 8g per L doesnt sound incredible but Id like to see their culture conditions.

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Think once high yield strains are isolated, then selective crossbreeding, coupled with further mutations on the progeny might help get there?

Is it easy to get claviceps to cross breed in culture as you suggest? That would be a real interesting option.

@Vesp

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I will consider it, but for me I think mutations is about the only option, and it will at least give me some good practice in biology lab type stuff. However, the input is very helpful and I will continue to research that out and look at the book you've linked too -- it might be more in reach than I believe - however, for someone that has done very little sterile work, very little culturing and is a noob to biology in general, it does seem to be a bit out there for me at least.

Im not saying its impossible to do it by random mutation just very difficult for a one man show is all., before you make the financial and personal commitment its best make damn sure you are on the right track and no easier option exists. The beauty of genetic engineering is that most of it is enzymatic and there are fewer cultures going on so a lower overall risk of contamination. Its actually amazingly straightforward. The aseptic technique is something you will master fairly quickly.

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What you are saying is that the tell that alkaloids are present, but not what type?

Exactly

Some comments from a brief scan of that document (full .pdf attached for reference) is that they seemed to suffer yield losses when they regenerated the cell walls of the protoplasts, they had access to whatever strain they wanted and could pick and choose them for their lifecycle stages in vitro, they have low regeneration efficiency so you need the technique to be tight and they had their choice of mutagens. If you are going to follow the protocols can you get the same mutagen by any chance?

That said even a 500mg per L culture is yielding a serious ammount of alkaloids given. Under freezing the microorganism seems to last years. They even briefly mention a mutant that produces 1100mg /L per 12 days so that should be the target... ie follow their suggestions of media and mutagen to obtain that baby :)A key to experiments such as this is organization in the beginning you need to plan this out like a bank robbery

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Also i don't think it would be that difficult to screen for thousands of mutations.
perhaps I am looking at this the wrong way.. but I imagine that if a mutagen is added to a liquid culture of ergot (LC) and allowed to incubate a bit, than a tiny bit of the mutants were smeared on a hundred or so re-usable petri dishes - hopefully giving many different strains on the ergot in each petri dish, you could find a few that give higher alkaloid content, especially since you could use the few hundred petri dishes several times. say 100 (petri dishes) x 5 (re-using the 100 dishes 5 times) x 10 (different colonies per petri dish) = 5000 different strains.

They used spectrophotometric assays to determine the alkaloid production. It is difficult by the naked eye to tell the difference between say dark blue and slightly darker blue whereas spotting dark blue against a light blue control is simple. The drop out rate for their mutations was pretty high.

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5000 different strains isn't that much at all considering how many genes there are -- however, each strain would likely contain many different mutations (I'd make sure of it Wink), but considering how many genes must play a roll in making and regulating the alkaloid production and content it seems like the alkaloid production could be increased via several ways, not just increased expression of a single gene - though again, I could be wrong. Think about how many things can affect the height of a human however - if the goal is to make a small human, it may not be only effecting the production of growth hormone, but also the protein which it actives, the ease of calcium uptake (i.e can't grow with limited bone growth) and many other factors I bet.

Relative to the genome 5,000 is not a lot but at say twenty minutes of your time each it piles up fairly rapidly. The number of genes required for increased production may actually not be that large, it depends on the metabolic pathway complexity -i.e. how many biosynthetic steps are required to produce the alkaloid. Its a lot easier if the organism is fed a metabolite that requires only one enzymatic digestion but there may be less than ten steps depending on what you feed them. The mutations you make will be in the geno type not the phenotype so expression of mutations will number far fewer than actual mutations. This is akin to the human height thing say one parent was a dwarf and one was average height the child would be more likely to be average height if the average height gene was dominant.

Is the intention to rely on the van urk reagent and a bit of microscopy to identify the initial strain?

Vesp · « **Reply #19 on:** July 29, 2010, 07:30:21 PM »

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Think once high yield strains are isolated, then selective crossbreeding, coupled with further mutations on the progeny might help get there?
Is it easy to get claviceps to cross breed in culture as you suggest? That would be a real interesting option.

I don't think ergot can cross breed in culture, can they? Seems like you'd have to infect rye plants at the right time with the medium and try to get their spores - than mix the spores from the different ergot strains in order to get them to technically breed. I assume they can breed in a similar fashion as to how magic mushrooms do?

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The beauty of genetic engineering is that most of it is enzymatic and there are fewer cultures going on so a lower overall risk of contamination. Its actually amazingly straightforward. The aseptic technique is something you will master fairly quickly.

It sounds like you have done this before... I think you may be right that random mutations are not going to be easy, but when it comes to the genetic engineering, I would need to use electrophoresis, buy probably expensive restriction enzymes and all sorts of stuff to isolate or double the wanted genes, Correct?

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you are going to follow the protocols can you get the same mutagen by any chance?

I hope so, I am still trying to work this all out.

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They used spectrophotometric assays to determine the alkaloid production. It is difficult by the naked eye to tell the difference between say dark blue and slightly darker blue whereas spotting dark blue against a light blue control is simple. The drop out rate for their mutations was pretty high.

I have a very old high school spectrophotometer that I believe only needs a light to work. Not sure if I can effectivly use it or if it is of a high enough quality for this, but using surplus lab supplies I do not personally believe that getting a better spectrophotometer is going to be very difficult... Good idea. I am not to sure how I could effectively use this when the organisms/alkaloids are on a petri dish, however.

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Is the intention to rely on the van urk reagent and a bit of microscopy to identify the initial strain?

Mostly, and other reagents that change colors based on alkaloids.

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Not one colony derived from protoplasts
regenerated without previous mutagenic treatment
of growing mycelia showed increased ergot
peptide synthesis. Frequencies of mutants with
increased levels ranged between 1 and 3%, with
no significant preference for either of the two
mutagens or media. All high-yielding mutants
showed a violet-brown pigmentation and an in-
creased tendency toward sclerotia-like morphol-
ogy compared to the parent strain, which is
white with slender vegetative hyphae and rela-
tively low fat content (data not shown). None of
the high-yielding strains listed in Table 1 has lost
its increased capacity of alkaloid formation with-
in the last 2 years. Several strains (e.g., 1013)
showed sectoring when grown as giant colonies,
whereas others (e.g., 1029) never did. Strains
with sclerotia-like morphology were kept on T 2
under liquid paraffin at -32°C. No loss of viabili-
ty was observed under these conditions after
storage for at least 1.25 years.
ATCC

I thought this was useful -- perhaps the darker colonies will contain higher alkaloid concentration. A problem of the ergot changing color though is that it screws up the tests that I was planning to rely upon to test for the conc. of alkaloids.

Author Topic: Mutating Organisms. How? (Read 477 times)

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