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Author: Subject: Yeast plasmid max size
Tsjerk
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[*] posted on 14-12-2018 at 15:26


Quote: Originally posted by Jackson  


Also i dont need a large ammount because i want to test if its bioactivity is similar to that of melatonin.

[Edited on 12/14/2018 by Jackson]


For that you need large amounts, large amounts compared to what you will be able to extract from the yeast. This point I tried to make when talking about the difference between production and mass spec conditions. What you think are small amounts... are orders of magnitude bigger than what you will be able to make and what was shown in the article.



[Edited on 14-12-2018 by Tsjerk]
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phlogiston
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[*] posted on 14-12-2018 at 15:32


Jackson, your basic idea is not bad.
It is just that from the questions you are asking it is obvious that you haven't got the slightest idea of the magnitude of the challenge.
Things like ampicillin resistance for selection of transormants is extremely basic stuff, you are missing the actual problems you are facing.

Also, your notion of 'small amounts' is very different from what you can realistically expect to make by this method.
If you are planning to 'assay bioactivity' in the way that I think you are, you'll need many milligrams. But yields would probably be in the ng - ug range. (And it would be in a very dilute solution so you'd have to purify and concentrate the product. But that is a minor challenge after you made the yeast).

Tsjerks assessment that it would cost several 100's of thousands of dollars to do this is very true. Even with that kind of money and a professional lab it would still be a risky project that has a high chance of failure.




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Tsjerk
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[*] posted on 14-12-2018 at 15:33


Quote: Originally posted by Jackson  
If i end up attempting this i plan to use an ampicillin resistance gene in the plasmid to select for the yeast that took in the plasmid. This way i can use a small amount of plasmid material and just grow the yeast with the plasmid inside them to get a larger ammount of modified yeast without using a large ammount of plasmid material.

If you would believe a graduated molecular biologist... you don't need just a small amount when transforming yeast. Definitely more than you think; multiple micro-grams.

Edit: As I already said; this is PhD stuff. It took me years to master this stuff and I still didn't.

[Edited on 14-12-2018 by Tsjerk]
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DoctorOfPhilosophy
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[*] posted on 14-12-2018 at 17:25


Quote: Originally posted by Jackson  
This way i can use a small amount of plasmid material and just grow the yeast with the plasmid inside them to get a larger ammount of modified yeast without using a large ammount of plasmid material.

Also i dont need a large ammount because i want to test if its bioactivity is similar to that of melatonin.


One of the many reasons you might want to try in bacteria first is because indeed, you don't need a large amount of plasmid to try a single transformation. Maybe 100 ng (at most). Of course if it doens't go well you have to try again, and considering that you might purchase 250 ng from the supplier (IDT in this example), that only gives you 2.5 tries. That's not including duplicates or triplicates which are usually a good idea to compensate for mistakes or bad luck.

Now the real reason why people put their plasmids into bacteria even if they're working with yeast or higher organisms, is because it's a nice stable way to store plasmids that can readily create more plasmids. It's a lot cheaper to transform some 10 dollar e coli cells than to order plasmids 10 times. And if you try to store the plasmid in the yeast cell then you will have all kinds of drawbacks if you want to make more or extract ("prep") it. Basically it won't multiply fast and it won't be stable, and it may or may not hold well in the freezer, especially a home freezer which you open all the time and put large warm things into.

Also, it's worth considering that 5-Methoxytryptamine is only 30 bucks on sigma, so buying it might be a faster way to research its pharmacodynamics.

[Edited on 15-12-2018 by DoctorOfPhilosophy]
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Jackson
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[*] posted on 14-12-2018 at 19:18


Oh, if it’s that cheap on sigma and it’s food grade then I guess I don’t need to try and attempt this whole thing.
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