NEMO-Chemistry
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How do you make yeast glow?
My plant cell experiment is not going too well, things might pick up now I have incubator space with decent lights in.
Anyway, having read up on protocols, i am still a bit lost with yeast.
How do I actually choose the site I want the FGP to bind too? This should be really simple shouldnt it. There is alot of info on using heat or
electric to align then absorb plasmids etc, but how and where do i get them from and which ones?
I dont have a particular gene preference, what I want is yeast to do their thing and glow under UV, and the small matter of making them antibiotic
resistant.
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Tsjerk
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You don't have to couple the fluorescent protein to anything, just let them make free GFP, it will be soluble in the cytoplasm.
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Metacelsus
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Well, you do need to couple the gene itself to a promoter, but as Tsjerk stated free GFP will be sufficient. If you want a more advanced (and
interesting) experiment, you could try expressing genes from the luciferase pathway in order to have the yeast glow all the time (not just under UV).
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NEMO-Chemistry
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Quote: Originally posted by Metacelsus  | | Well, you do need to couple the gene itself to a promoter, but as Tsjerk stated free GFP will be sufficient. If you want a more advanced (and
interesting) experiment, you could try expressing genes from the luciferase pathway in order to have the yeast glow all the time (not just under UV).
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Thanks that gives me a start point to study. Glowing all the time would be good.
The reason i want to do this is more complicated than i am currently able to explain. Once i understand how it works better, i will repose the
question in a more coherent fashion.
How or rather where do I start looking, in order to understand how to express a specific gene? I know this all sounds like i am a muppet, but i have
read a massive amount on this, and the main bit i am missing in my understanding, is exactly how you go about expressing the gene.
Alot of the other bits i understand, but the mechanics of how its done i dont know.
For example I know how to fuse plant cells etc, and i know how to get a plant cell to take in another nucleus, what i dont know the mechanics of, is
how do you actually turn a gene on or off?
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NEMO-Chemistry
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Thank you by the way, the answers are helpful.
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phlogiston
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Briefly: the stretch of DNA just before the start codon of the gene contains special sequences that determine when and to what extent the gene is
expressed.
This stretch of DNA that determines the level of expression is called the 'promotor'.
You can use a promotor from another gene that has the characteristics that you desire. Perhaps you want your gene to be most strongly expressed when
the yeast is fed sugar? Maybe you just want it to be 'on' all the time? Maybe you want the strongest promotor known to get maximal fluorescence?
Construct/buy/use a plasmid that has a promotor with the properties that you want.
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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Metacelsus
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I would suggest taking a look at this site: http://blog.addgene.org/plasmids-101-yeast-vectors
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unionised
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OK, I will see what I can do about that.
:-)
How do you make yeast glow?
Heat to 600C in a reducing atmosphere
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NEMO-Chemistry
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What would i do without you? Answers on a postcard only please.
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NEMO-Chemistry
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Thanks again chaps. Slowly the gaps are filling up!!
In a nut shell, I want one type of yeast to glow, then when/if it mates with another that dosnt glow, I want the F1 to glow.
I would rather gather the equipment to do the plasmids, than buy them.
Yeast are my start point because i have worked with them alot. Once I have the technique mastered, I will move onto my target organism.
[Edited on 15-1-2018 by NEMO-Chemistry]
[Edited on 15-1-2018 by NEMO-Chemistry]
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Tsjerk
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Don't forget the ribosomal binding sequence (RBS)! You wouldn't be the first.
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NEMO-Chemistry
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Well something is badly wrong somewhere! For a start why is GFP so expensive for what it is?
Adding it to yeast culture didnt work.......I wont detail everything, i am missing something somewhere. I have read these protocols over and over, not
just papers but complete books. I have read many books actually, many different protocols for these kind of techniques.
Some of the more obscure and amateur ones have work the best, but i am now pretty sure i am missing some really basic 101 stuff, or snip of info
somewhere. Really frustrating!
Other issues with the plants I got over easy enough, like dissecting one side of the membrane on a leaf to expose the cells, alot of books talk of
slicing up, or literally peeling with a knife.
Turns out sellotape is your best friend for this! dead simple and really effective. Right one more read up and try with this and yeast.
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Metacelsus
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| Quote: Originally posted by NEMO-Chemistry | Well something is badly wrong somewhere! For a start why is GFP so expensive for what it is?
Adding it to yeast culture didnt work.......I wont detail everything, i am missing something somewhere. |
Wait, do you mean you added the GFP protein to yeast? Of course this won't work; you need to add a plasmid (DNA) containing the GFP gene so that the
yeast make GFP. A GFP plasmid should be pretty cheap to obtain.
More details would be appreciated.
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Tsjerk
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I'm pretty sure NEMO didn't add the protein. Although a detailed description would be useful for reviewing purposes.
Vector DNA is expensive, but often PhD students or post-docs are willing to give you a small aliquot if you ask nicely. I would give you anything if I
were still in the lab. I can imaging you can get standard parts from iGEM competers if you ask them during "the season"
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NEMO-Chemistry
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Well i tried various protocols, mainly based on heat shock. I brought FGP plasmids and was told they were the idea for my yeast strain.
I need to go all the way back to the start and try again, to me the plasimids containing FGP or really expensive, i am now broke until the summer
again. I have contacts for most things, but this is a gap in my contact list. I even have a electroporation machine now but no curvetes yet.
Getting equipment donated from universities seems easy, but samples of organisms etc are getting harder. Messing up £100 of agar dosnt help, i
brought some in bulk to cut costs, when i opened the foil pouch thing i pressed to hard and the bottom popped open!!
pile of it on a dirty garage floor  . Just one of those week this week. I will hunt
through the books and pick a single protocol to follow. Anyone got one to recommend? If I cant get this working with yeast, then i have no chance
getting it to work for what i really want to do with it.
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j_sum1
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Thread Split
23-1-2019 at 17:38 |
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