Growing out yeast spheroplasts - (Jun/10/2008 )
Hello,
I am working on creating yeast spheroplasts (by lytic digestion of the cell wall) so that I can treat them with a compound that is blocked by the presence of the cell wall. Eventually, I'd like to be able to grow these spheroplasts out for several generations. I'm working on a protocol to 1) grow out the spheroplasts, and 2) demonstrate that the outgrown cells came from spheroplasts rather than any lingering intact yeast cells in the culture. Any suggestions would be much appreciated. I'm especially having trouble keeping the spheroplasts alive long enough to treat them and plate them, and this is important because I will eventually need to demonstrate that the compound is killing them (as opposed to their just dying off on their own). Thanks in advance!
-AW
-Anna Jean-
QUOTE (Anna Jean @ Jun 10 2008, 07:34 AM)
Hello,
I am working on creating yeast spheroplasts (by lytic digestion of the cell wall) so that I can treat them with a compound that is blocked by the presence of the cell wall. Eventually, I'd like to be able to grow these spheroplasts out for several generations. I'm working on a protocol to 1) grow out the spheroplasts, and 2) demonstrate that the outgrown cells came from spheroplasts rather than any lingering intact yeast cells in the culture. Any suggestions would be much appreciated. I'm especially having trouble keeping the spheroplasts alive long enough to treat them and plate them, and this is important because I will eventually need to demonstrate that the compound is killing them (as opposed to their just dying off on their own). Thanks in advance!
-AW
I am working on creating yeast spheroplasts (by lytic digestion of the cell wall) so that I can treat them with a compound that is blocked by the presence of the cell wall. Eventually, I'd like to be able to grow these spheroplasts out for several generations. I'm working on a protocol to 1) grow out the spheroplasts, and 2) demonstrate that the outgrown cells came from spheroplasts rather than any lingering intact yeast cells in the culture. Any suggestions would be much appreciated. I'm especially having trouble keeping the spheroplasts alive long enough to treat them and plate them, and this is important because I will eventually need to demonstrate that the compound is killing them (as opposed to their just dying off on their own). Thanks in advance!
-AW
Is this a new protocol you are developing or is it a standard one? I have not heard of spheroplast culture, but then, I don't do yeast work!
-cellcounter-
QUOTE (cellcounter @ Jun 10 2008, 01:40 PM)
QUOTE (Anna Jean @ Jun 10 2008, 07:34 AM)
Hello,
I am working on creating yeast spheroplasts (by lytic digestion of the cell wall) so that I can treat them with a compound that is blocked by the presence of the cell wall. Eventually, I'd like to be able to grow these spheroplasts out for several generations. I'm working on a protocol to 1) grow out the spheroplasts, and 2) demonstrate that the outgrown cells came from spheroplasts rather than any lingering intact yeast cells in the culture. Any suggestions would be much appreciated. I'm especially having trouble keeping the spheroplasts alive long enough to treat them and plate them, and this is important because I will eventually need to demonstrate that the compound is killing them (as opposed to their just dying off on their own). Thanks in advance!
-AW
I am working on creating yeast spheroplasts (by lytic digestion of the cell wall) so that I can treat them with a compound that is blocked by the presence of the cell wall. Eventually, I'd like to be able to grow these spheroplasts out for several generations. I'm working on a protocol to 1) grow out the spheroplasts, and 2) demonstrate that the outgrown cells came from spheroplasts rather than any lingering intact yeast cells in the culture. Any suggestions would be much appreciated. I'm especially having trouble keeping the spheroplasts alive long enough to treat them and plate them, and this is important because I will eventually need to demonstrate that the compound is killing them (as opposed to their just dying off on their own). Thanks in advance!
-AW
Is this a new protocol you are developing or is it a standard one? I have not heard of spheroplast culture, but then, I don't do yeast work!
Not standard, but not completely new either. I have heard from a few people and from the literature (old literature... from the early 80's when spheroplast formation was discovered) that it is possible and is very occasionally done. Once they are in culture, they should regrow their cell walls and begin dividing normally. My lab just needs to have them as spheroplasts long enough to treat them (the treatment compound doesn't work on intact yeast) and eventually select for spheroplasts that are resistant to a particular compound. We need to culture them, because we want to cultivate that strain of resistant yeast for later genetic analysis. I'm not sure whether this is a new technique... but there isn't a whole lot of information out there on it, in either case.
-AW
-Anna Jean-
QUOTE (Anna Jean @ Jun 10 2008, 10:39 AM)
Not standard, but not completely new either. I have heard from a few people and from the literature (old literature... from the early 80's when spheroplast formation was discovered) that it is possible and is very occasionally done. Once they are in culture, they should regrow their cell walls and begin dividing normally. My lab just needs to have them as spheroplasts long enough to treat them (the treatment compound doesn't work on intact yeast) and eventually select for spheroplasts that are resistant to a particular compound. We need to culture them, because we want to cultivate that strain of resistant yeast for later genetic analysis. I'm not sure whether this is a new technique... but there isn't a whole lot of information out there on it, in either case.
-AW
-AW
In that case, let me suggest some possible methods:
You transform the spheroplats with GFP or some other marker (lac-z, antibiotics), only the spheroplasts should be transformed with GFP and not intact cells, then treat the transformed mixture with your compound and plate them. If you see less number of GFP positive colonies/cells in test Vs. vehicle treatment, you have proved your point.
You may also look for a chemical that works for tracking the cells..eg. some fluorescent dyes that would not stain the intact yeast cells but stain spheroplasts.
The spheroplasts should grow just as well for an optimized compound exposure time (if they don't Vs. vehicle, again, you have proved your point) but I don't think there will be many generations in either case, because they will grow cell wall within one generation, I guess.
-cellcounter-
I'm familiar with the generation of spheroplasts but have no idea how that condition might be maintained. Conceptually - it's difficult to see how they could "grow" as a yeast culture - budding is a cell-wall dominated phenomenon and even that presumes you'd find a way to stop cell wall synthesis.
How have you designed your experiment?
-jorge1907-
Since yeasts are easy to manipulate, may it be possible to use a yeast mutant with defect cell wall assembly? I have there no special mutant in mind but someone else may help. Which component of the cell wall is inhibiting your compound?
-jazim-
"Yeast" is a direct function of cell wall integrity - bud requires cell wall integrity. The concept would require spheroplast population being established, knockouts/inhibitors of cell wall polysaccharide (glucans, mannans, chitin) effectively applied in an osmotically stabilizing environment. Doubt you'd get "growth" - reproduction of cellular units.
-jorge1907-
QUOTE (jorge1907 @ Jun 10 2008, 05:56 PM)
I'm familiar with the generation of spheroplasts but have no idea how that condition might be maintained. Conceptually - it's difficult to see how they could "grow" as a yeast culture - budding is a cell-wall dominated phenomenon and even that presumes you'd find a way to stop cell wall synthesis.
How have you designed your experiment?
How have you designed your experiment?
We are not trying to stop the growth of the cell wall once we have plated the spheroplasts. Rather, we are treating them while they are spheroplasts and then allowing regrowth afterwards. The experiment is attempting to demonstrate that certain strains of yeast will be resistant to this compound while others won't. Thus, the experimental set up involves making spheroplasts from a particular yeast strain, treating those spheroplasts with the compound, then plating the spheroplasts and allowing them to regrow (or not... whichever is the case). The difficulty is demonstrating that the regrowth plate originated from spheroplasts rather than left over intact yeast cells whose cell walls did not get digested in the process.
-Anna Jean-
QUOTE (cellcounter @ Jun 10 2008, 02:53 PM)
QUOTE (Anna Jean @ Jun 10 2008, 10:39 AM)
Not standard, but not completely new either. I have heard from a few people and from the literature (old literature... from the early 80's when spheroplast formation was discovered) that it is possible and is very occasionally done. Once they are in culture, they should regrow their cell walls and begin dividing normally. My lab just needs to have them as spheroplasts long enough to treat them (the treatment compound doesn't work on intact yeast) and eventually select for spheroplasts that are resistant to a particular compound. We need to culture them, because we want to cultivate that strain of resistant yeast for later genetic analysis. I'm not sure whether this is a new technique... but there isn't a whole lot of information out there on it, in either case.
-AW
-AW
In that case, let me suggest some possible methods:
You transform the spheroplats with GFP or some other marker (lac-z, antibiotics), only the spheroplasts should be transformed with GFP and not intact cells, then treat the transformed mixture with your compound and plate them. If you see less number of GFP positive colonies/cells in test Vs. vehicle treatment, you have proved your point.
You may also look for a chemical that works for tracking the cells..eg. some fluorescent dyes that would not stain the intact yeast cells but stain spheroplasts.
The spheroplasts should grow just as well for an optimized compound exposure time (if they don't Vs. vehicle, again, you have proved your point) but I don't think there will be many generations in either case, because they will grow cell wall within one generation, I guess.
The idea of transformation occurred to me, but it would be a bit tricky because its imperative that the genetic material of the yeast remains unaltered. Coding for antibiotic resistance or some other marker could very possibly make a difference in the genetic analysis we want to do after we have developed a strain of compound resistant yeast. A plasmid vector might work, in this case, because I don't think whatever marker it coded for would persist over several generations, but I'm still reluctant to introduce any new genetic material to the cells.
The flourescent dye idea I really like... I think I'll look into that. There is a strong possibility that there is something I can find that binds exclusively to cell walls.
Thanks so much!
-Anna Jean-
If you've a mixed population - why do you think you've a subpopulation free of cell walls?
Perhaps you could enrich by differential centrifugation but trying to differentiate response without investing in a clean prep doesn't sound so useful.
-jorge1907-