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 AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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 Creating Genetic Hybrids Through Electroporation?
#8993135 - 09/27/08 11:13 AM (2 months, 6 days ago) |
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As I look for different ways to produce genetic hybrids, I came across the method known as electroporation. Initially to attract donor DNA, the recipient fungi cells are initially treated with a chemical flocculant. Next, an electric field is applied to the recipient cells suspended in liquid culture. When exposed to an electric field, the naturally occurring pores in the cell wall widen and as a consequence pull in the donor DNA.
To test and see if the recipient cells picked up the donor DNA, one doesn't have to take the cells all the way to the fruiting stage. One can grow the cells in liquid culture, harvest the cells, mechanically digest the cells, and run the filtrate through a chromatography separation procedure. In comparing the chromatograms to untreated cells, to treated cells, and finally to the donor cells, one can determine if the treated cells are now producing proteins unique to the donor fungi cells. If the chromatograms show that the recipient cells are now producing foreign protein, they can be taken through the fruiting stage to see if the foreign protein production is observable at the macroscopic level.
Has anyone attempted this before?
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 RogerRabbit
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 Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#8993200 - 09/27/08 11:27 AM (2 months, 6 days ago) |
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Since most members here don't have access to DNA testing, I seriously doubt it.
RR
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denger
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Re: Creating Genetic Hybrids Through Electroporation? [Re: RogerRabbit]
#8993316 - 09/27/08 11:48 AM (2 months, 6 days ago) |
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I have the same suspicion as RR.
This goes back to my question earlier about a system to select the results of a cross.
Having such a system would simplify testing new hybridization methods and procedures significantly.
See the thread here:
Hybrid selection methods
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Dennis, in Love with Fungi
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#8995067 - 09/27/08 06:15 PM (2 months, 6 days ago) |
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Quote:
denger said:
Having such a system would simplify testing new hybridization methods and procedures significantly...
Chromatography is the best and most accessible method that I'm aware of. If the initial chromatogram of a mushroom isolate is not definitive enough due to the lack of enough distinguishable bands, one can use selective proteases. Proteases like pepsin, trypsin, and chymotrypsin, cleave amino acid bonds at very specific sites. Due to their specificity, when a mushroom isolate that may initially show three distinct bands is selectively digested with pepsin, the initial three bands may break into four or more bands. Pepsin is selective in that it will always cleave amino acid bonds at the C-terminal of tyrosine. Due to its consistent selectivity, a new pattern of bands will emerge in the chromatogram that will be specific to the way pepsin digests the mushroom isolate.
However, if your mushroom isolate doesn't have any tyrosine in its structure, you will still see the original three bands show up in the chromatogram. If this happens you could try trypsin, that selectively cleaves the amino acid bonds between lysine and arginine. Chymotrypsin on the hand cleaves amino acid bonds next to phenylalanine.
If you have trouble getting a hold of these proteases, you could try and isolate your own pepsin from vegetable sources like papaya.
I really don't think it's that complicated. You really don't even need to know the amino acid sequence of the proteins you're working with. All you need to know is what's really happening in each procedure. If you're patient and like puzzles, you'll like chromatography. It really isn't that difficult, just really tedious work.
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denger
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#8995435 - 09/27/08 07:20 PM (2 months, 6 days ago) |
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I am very familiar with the procedure, but it will only allow you to examine limited number of potential hybrid candidates.
How many of them can you realistically check?
Lets say you need 10mg of material to examine. That means you need to grow each potential colony to a diameter of 3cm. Thats at most 3 colonies on a standard dish. So, even if you grow 100 dishes you will only have material from 300 potential clones.
Next, you will need to run at least 300 chromatograms.
It is not unusual to have a very low rate of success when doing hybridizations of incompatible organisms. Maybe on the order of 1 in 10^6 - 10^8. You will have no hope of growing that many colonies and examining them with this method.
A selective system is a must in such scenario.
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Dennis, in Love with Fungi
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#8995561 - 09/27/08 07:45 PM (2 months, 6 days ago) |
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Quote:
denger said:
I am very familiar with the procedure, but it will only allow you to examine limited number of potential hybrid candidates.
How many of them can you realistically check?
Lets say you need 10mg of material to examine. That means you need to grow each potential colony to a diameter of 3cm. Thats at most 3 colonies on a standard dish. So, even if you grow 100 dishes you will only have material from 300 potential clones.
Next, you will need to run at least 300 chromatograms.
It is not unusual to have a very low rate of success when doing hybridizations of incompatible organisms. Maybe on the order of 1 in 10^6 - 10^8. You will have no hope of growing that many colonies and examining them with this method.
A selective system is a must in such scenario.
You're incredibly correct. There are definitely limitations to the process. It's really repetitive and tedious work. Not only that, there are no guaranteed results. Even if one were to successfully make a hybrid from edible incompatible species, the resulting mushroom may not look appetizing at all. It may simply look like a tumorous growth of sorts. Or, at the other end of the spectrum of possibilities, your chromatogram confirms the production of foreign protein only associated with the donor DNA, but the mushroom doesn't look or taste any different from the original species.
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denger
Mycelium keeper
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#8995633 - 09/27/08 08:01 PM (2 months, 6 days ago) |
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Right, so why not look for (or create) a system that lets you overcome these limitations?
Such systems are well described and widely used on other organisms.
All we need is to find/create strains that are deficient (require an additive to grow) or resistant to some chemical (such as antibiotics in bacteria).
Here is an example: you have strain A, which say cannot grow unless you have chemical "a" in the media. You also have strain B, which cannot grow on a media that does not have chemical "b". Neither one of these strains will grow on a media that does not contain neither a nor b. If you mix these two strains, and plate on this media, nothing will grow. However, if you use electroporesis, and plate the results on such media, then some certain hybrids between the two will survive. You can plate literally hundreds of millions individual mycelial fragments, and will only see the hybrids grow. No additional work required.
The beauty and simplicity of such a system allows to make numerous hybridization experiments with very little effort or investment.
I think people who are interested in hybridizing mushrooms should collectively start working on creating such a system.
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Dennis, in Love with Fungi
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#8995876 - 09/27/08 09:04 PM (2 months, 6 days ago) |
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Quote:
denger said:
Right, so why not look for (or create) a system that lets you overcome these limitations?
Such systems are well described and widely used on other organisms.
All we need is to find/create strains that are deficient (require an additive to grow) or resistant to some chemical (such as antibiotics in bacteria)...
Interestingly enough, I asked a similar question to my organic chem professor and my microbio professor. They made similar statements but it essentially boils down to if you're looking for a change in genetics, one needs to tailor the experiments to probe at the genetic level, i.e. isolating DNA and performing electrophoretic separations.
Creating environments that selects out strains based on a strains ability to survive with or with out certain nutrients is not conclusive unless one has a strain that clearly cannot survive with out a certain nutrient. An example that my microbio professor gave was that finding an organism that is deficient in some way is really difficult to do. Switching the growing medium to something the fungi does not normally grow on and inoculating it with various fungal hybrids may not automatically select a genetically different strain. The strains that do grow on the foreign substrate may simply be expressing its innate ability to adapt to a different growing medium.
Creating a strain that is deficient in some way may be easier to do. I talked to this geneticist back in junior high about this. The way to proceed is to get a sample of the organism and dry it as thoroughly as possible. In the case of mushrooms, the best way is to proceed is to use the dry spores or lyophilized mycelium and expose them to incredibly high doses of ionizing radiation. The samples have to be absolutely dry because when water or hydrated compounds are exposed to ionizing radiation, free radicals tend to be produced that cause cellular damage and not genetic damage. Next, the spores need to be plated on a nutrient rich medium and then isolated to see which ones express a nutrient deficiency. The tricky part though is that lower lifeforms like bacteria and I suppose fungi too have incredible efficient genetic reparative mechanisms. Therefore isolating a strain that has a nutrient deficiency is one thing, but finding one that stays deficient is another thing.
The major problem though in going this route is the need for a source of incredibly high gamma ray radiation. The source that I used in my science fair experiment was a cobalt 60 source found at the local university. The radiation it emitted was so strong that it needed to be kept submerged in a 25 foot deep pool of water. It was so strong that when they turned off the lights in the laboratory, the bottom of the pool glowed blue. Even though I was trying to create genetic mutations in vegetable seeds, due to the size of the seeds, the radiation dose that was used ranged in the 500 mega rad range. Keep in mind that a lethal dose for the average human is only 500 rads. To create mutations in spores that are much much smaller than my radish seeds, the dose will be significantly higher, possibly in the giga rad range.
Sorry for rambling, never the less, I guess the reason why no one has created fungal strains that have nutrient deficiencies is because the materials needed can only be found at the university level. Not only that, much of the research that gets funded is funneled towards bacterial and viral research. Not much money is being sent to research in mycology.
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Aeolus1369
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#9074345 - 10/14/08 12:44 AM (1 month, 20 days ago) |
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Can you cite where you got that passage? I've only ever used electroporation with bacterial and mammalian cells
To my knowledge, electroporation can only be used for moderately sized pieces of DNA and if I understand you, you're talking about electroporating whole nuclei of genomic DNA. The odds of getting something like that into another cell intact is probably impossible. If you had some way to select for successful transformants, you could at least grow up the hybrid from a single cell, but without selection your signal to noise ratio is going to be null for all intents and purposes.
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AdoreChampignons
Mycophilic One
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Posts: 313
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Re: Creating Genetic Hybrids Through Electroporation? [Re: Aeolus1369]
#9079077 - 10/14/08 10:51 PM (1 month, 19 days ago) |
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Quote:
Aeolus1369 said:
...The odds of getting something like that into another cell intact is probably impossible. If you had some way to select for successful transformants, you could at least grow up the hybrid from a single cell, but without selection your signal to noise ratio is going to be null for all intents and purposes...
Yes, what you're saying is quite true. Molecular size is an issue as well as the selection process. I've done thought experiments on how I would carry out the process and I came up with the same problematic issues as you have just stated. To solve the size issue, I've thought of partially digesting the DNA and then trying the electroporation process. I have almost all the equipment already. However, whatever I do, I know it's going to be a tedious process. I need to ask a few more people about how I can increase the the "signal to noise ratio."
You are also right about the usage of electroporation being more studied in mammalian and bacterial systems. Since I have practically everything I need except a good selection process, I'm still in the planning stage than an executable one.
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Aeolus1369
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#9079200 - 10/14/08 11:19 PM (1 month, 19 days ago) |
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Quote:
AdoreChampignons said:
To solve the size issue, I've thought of partially digesting the DNA and then trying the electroporation process.
Hmm, interesting thought. I don't know that much about fungal cell biology...do you know how fungal cells treat foreign DNA? Obviously it has some sort of mechanism for recognizing and tolerating DNA from mating partners but if it's in fragments it might think it's foreign and just chew it up. Also what about homologous recombination if you were to say introduce via electroporation a different allele of a particular gene? You might end up with a monokaryon hybrid rather than a dikaryon if it recombines similar to how yeast does it.
Are there any sort of viral-mediated DNA delivery systems in fungi? My impression was viruses don't work too well because of the thick cell wall but I'm sure there's something out there that can transduce them.
Would appreciate any reference you could throw my way!
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Creating Genetic Hybrids Through Electroporation? [Re: Aeolus1369]
#9090445 - 10/17/08 07:34 AM (1 month, 17 days ago) |
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Quote:
Aeolus1369 said:
Quote:
AdoreChampignons said:
To solve the size issue, I've thought of partially digesting the DNA and then trying the electroporation process.
Hmm, interesting thought. I don't know that much about fungal cell biology...do you know how fungal cells treat foreign DNA? Obviously it has some sort of mechanism for recognizing and tolerating DNA from mating partners but if it's in fragments it might think it's foreign and just chew it up. Also what about homologous recombination if you were to say introduce via electroporation a different allele of a particular gene? You might end up with a monokaryon hybrid rather than a dikaryon if it recombines similar to how yeast does it.
Are there any sort of viral-mediated DNA delivery systems in fungi? My impression was viruses don't work too well because of the thick cell wall but I'm sure there's something out there that can transduce them.
Would appreciate any reference you could throw my way!
Yes, I hear what you're saying. I don't know much about fungal cell biology. I've been trying to find mycologists at the local university here. However, most of them are bacteriologist and virologist. Most of them only know how to kill fungi rather than grow them. Most of them say that there isn't much research money in growing mushrooms, therefore many grad students rarely focus their studies in that area. However, I'll keep on asking.
In the area of viral-mediated transmission of DNA fragments, there are possibilities in that area. One microbiologist told me with great confidence that bacteria, fungi, and viruses have been around much longer than we have. There is very likely a virus that can insert genes into fungal tissue. However, finding it is one issue and learning about its biology is another issue. Also viruses tend to be incredibly specific as to the type or strain of the host it attaches itself to.
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Plasmid
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#9096007 - 10/18/08 12:20 PM (1 month, 16 days ago) |
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I am only familiar with electroporation as applied to bacteria, in which case bacteria can pick me up (a joke: electroporation can be used to insert plasmids or expression vectors into bacteria). I don't know how this is done with yeast, which are fungi.
I would think that using electroporation would not allow you to do more than to insert small plasmids into fungi, which may or may not integrate into the host chromosome. I really don't know how stable a plasmid would be in an organism that produces different types of tissues. Even if it was stable, you'd really be producing a fungus with an expression vector and not really a "hybrid" species (though this is debatable, because I think the existence of plasmids blurs the notion of a species). If you could get the DNA to integrate into the chromosome, then you could achieve what you want.
I think that the approach most likely to be successful would be to use Agrobacterium tumefaciens to insert DNA into fungi. A. tumefaciens is commonly used for this purpose (the tumour inducing plasmid in A. tumefaciens usually inserts T-DNA into plants to cause the plant to produce nutrient - modern genetic engineering experiments replace the T-DNA with specific, desired genes). There'd be a lot of little details you'd need to know:
how do you change the tumour inducing (Ti) plasmid's host specificity to get it to transport DNA into fungi?
What restriction endonucleases will you need to cut out the T-DNA and insert your own DNA? What gene would you want to insert in the first place?
The possibilities are pretty amazing, but in all practicality would be expensive to carry out. I am sure that a bright 4th year biochemistry student would have the theoretical know how (and probably some of the practical know-how) to carry this kind of experiment out, but even restriction endonucleases are expensive.
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Plasmid
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#9096025 - 10/18/08 12:26 PM (1 month, 16 days ago) |
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Quote:
denger said:
This goes back to my question earlier about a system
I'm guessing my discussion on this won't be helpful in practicality, but I still think it's interesting and would like to see what others think.
If you were looking to modify a particular trait, such as nutrient uptake, biosynthesis of an enzyme involved in psilocybin production, etc. and knew where the gene(s) involved were located, a simple endonuclease digestion could let you isolate the stretch of DNA you're looking for from an agarose gel. Universities often offer DNA sequencing services for quite cheap (I can get 300 bp sequences for about $30) and the advantage would be that it's unlikely anyone would be aware that you're growing shroomies. That way you could monitor genetic changes and select the hybrids you wanted.
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AdoreChampignons
Mycophilic One
Registered: 08/10/08
Posts: 313
Loc: Seti Alpha 5
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Re: Creating Genetic Hybrids Through Electroporation? [Re: AdoreChampignons]
#9096174 - 10/18/08 01:24 PM (1 month, 16 days ago) |
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A few of you were asking about the electrical source I'm planning to use. The electrical source is going to be a TENS unit. TENS is an acronym for Trans cutaneous Electrical Nerve Stimulation. Physical therapists and chiropractors use it to stimulate nerves in and around muscles associated with chronic pain. While the controls on the device offers many variations to its output, the strength of the electric field can also be controlled by the distance between the electrodes along with the electrical conductivity(electrical resistance due to ion concentration).
As for a selection process, I'm researching the use of innocuous florescent proteins. As a mycelial colony grows on nutrient agar it assimilates various nutrients, excretes metabolites, and as a consequence alters the composition of the agar with in its immediate vicinity. Assuming that mycelium that has picked up donor DNA may alter the agar composition differently from that of mycelium that has not picked up any donor DNA, this variance in agar composition may be visibly different under UV light if florescent proteins were incorporated into the nutrient agar.
However, before I even get to the stage of developing a selection process, I first need to determine if the electroporation procedure will even work. Testing for assimilation of foreign DNA via the production of foreign protein shouldn't be that difficult. TLC chromatography will do that. Never the less, there a number of variables that I'd like to explore. For example, would monokaryotic mycelium be more receptive to donor DNA than dikaryotic mycelium? Would starving the host mycelium make the pores in the cell wall less selective to what it allows in, there by making it easier to absorb donor DNA? Would a monokaryotic host be more receptive to DNA, or partially digested DNA, from a monokaryotic donor? There are quite a few questions to explore and answer.
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There is no such thing as a dumb question. There are just curious people trying to learn something new.
Edited by AdoreChampignons (10/18/08 01:50 PM)
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denger
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Re: Creating Genetic Hybrids Through Electroporation? [Re: Plasmid]
#9096270 - 10/18/08 01:54 PM (1 month, 16 days ago) |
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Quote:
Plasmid said:
I would think that using electroporation would not allow you to do more than to insert small plasmids into fungi, which may or may not integrate into the host chromosome.
There is no particular reason to use the whole plasmids with fungi. Unlike bacteria, Fungi have no mechanism to maintain plasmids through cellular generations, so they are worthless as a vector and would introduce additional genetic material, which is unnecessary.
Which brings me to the question if there is an appropriate vector for genetic manipulation of fungi?
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Dennis, in Love with Fungi
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fastfred
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#9123486 - 10/23/08 08:36 PM (1 month, 11 days ago) |
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Wow! A lot of BS and misinformation in this thread. Don't have the time to cover everything, but a few points...
A TENS unit isn't going to be powerful enough to do electroporation.
Electroporation will do noting to cells with a cell wall. You first have to prepare protoplasts (dissolve the cell wall), which can be a pain.
As mentioned you aren't going to be getting large pieces of DNA into the cell. If you chop it up with a restriction enzyme and it manages to make it into the cell it will simply be digested by nucleases.
You really need a selection system. Doing chromatography on tens of thousands of samples is foolish. What you're basically talking about is isozyme analysis, which you would do on an SDS/PAGE gel, not on a column. People don't use that for screening colonies, it's simply too low volume, expensive, and tedious.
What you need to do is use nutritional complementation (use auxotrophic mutants as parents). That is really the only way to do it. Generating auxotrophic mutants isn't hard at all. Just fry them with UV, or use chemical mutagens or radiation, whatever is handy. Then use a "filtration enrichment" method (google it) to select for auxotrophs.
As far as fusing your parents you'll need to prepare protoplasts and fuse them using PEG or electrofusion. Electrofusion really isn't worked out all that well and is way more of a PITA than PEG mediated fusion.
> Testing for assimilation of foreign DNA via the production of foreign protein shouldn't be that difficult.
Maybe if you had plenty of money and a lot of time on your hands. Usually you would just look for an altered phenotype/morphology. I don't see why you would bother with tedious testing when you can just look at the damn thing and see if it's morphology is altered.
> would monokaryotic mycelium be more receptive to donor DNA than dikaryotic mycelium?
For a large number of reasons monokaryotic mycellium is almost always used. The main reason is that genotypes are more easily expressed in haploid organisms.
> appropriate vector for genetic manipulation of fungi?
Agrobacterium is commonly used for transformation of fungi. There are a number of plasmid constructs with hpf (hygromyicin resistance) and GFP DNA in them. Your real problem is finding a promoter and terminator that will work in your species. Mixing and matching promoters from different species has met with pretty mixed results. Lots of times it fails to express proteins.
I suggest looking up "agrobacterium, agaricus" if you're interested in some interesting experiments. It really shouldn't be that expensive to make something like a GFP mushroom. You could simply get a ready-made plasmid with hpf and GFP on it, electroporate it into agro, and co-cultivate with mycelium, then just select for hygromyicin resistance.
-FF
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Plasmid
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Re: Creating Genetic Hybrids Through Electroporation? [Re: fastfred]
#9123747 - 10/23/08 09:18 PM (1 month, 11 days ago) |
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Quote:
fastfred said:
> Testing for assimilation of foreign DNA via the production of foreign protein shouldn't be that difficult.
Maybe if you had plenty of money and a lot of time on your hands.
. . . You could simply get a ready-made plasmid with hpf and GFP on it, electroporate it into agro, and co-cultivate with mycelium, then just select for hygromyicin resistance.
LOL. It's simple to get a commericially available plasmid, transform A. tumefaciens, etc. but not test for the presence of a specific protein?
Testing for the presence of a specific protein can be easy or difficult. It depends on the protein and on what's being done with it. Since it doesn't seem like the goal would be to overexpress the protein, it's probably not going to be as easy to find as a protein produced in E. coli with that is overexpressed. However, if you know something about the protein that you're looking for, which you should, then there's not any reason to think beforehand that it would be harder than looking for a morphology change or phenotype change. Plus, you'd better be damn sure that the phenotype change is the result of expression of the protein you're interested in, instead of being due to something else. On the other hand, if you know something about the protein you're interested in (like pI, MW, reactivity with a specific substrate, whatever), then you can get better evidence that you have what you want.
Quote:
Agrobacterium is commonly used for transformation of fungi. There are a number of plasmid constructs with hpf (hygromyicin resistance) and GFP DNA in them. Your real problem is finding a promoter and terminator that will work in your species.
In this scheme, are you talking about DNA integrating with the fungi's chromosome (I'm assuming you must be)?
Why bother using GFP at all, unless you're actually trying to visualize something in the cell?
Where are these plasmids available from? NEB? GE Healthcare? If you could provide a plasmid name, that'd be great.
Edited by Plasmid (10/23/08 10:13 PM)
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denger
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Re: Creating Genetic Hybrids Through Electroporation? [Re: fastfred]
#9123760 - 10/23/08 09:22 PM (1 month, 11 days ago) |
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Thank you, ff!
Finally, someone who knows what they are saying.
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Dennis, in Love with Fungi
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MycoAu
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Re: Creating Genetic Hybrids Through Electroporation? [Re: denger]
#9123891 - 10/23/08 09:45 PM (1 month, 10 days ago) |
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FF, could you post a paper or two (just the reference will work) of that PEG method you mentioned.
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