Do plasmids introduced via electroporation replicate with subsequent cell divisions?
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Cory Geesaman
Sep 19, 2017, 12:33:09 PM9/19/17
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The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
Rikke Rasmussen
Sep 19, 2017, 12:49:55 PM9/19/17
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In bacteria, at least, the plasmids are replicated with the cell cycle - it's how "we" make more plasmid. =)
On Tue, Sep 19, 2017 at 9:33 AM, Cory Geesaman <co...@geesaman.com> wrote:
The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
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Bryan Jones
Sep 19, 2017, 2:02:34 PM9/19/17
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The plasma requires an origin of replication that works with the host. Different plasmids tend to be maintained at different number copies per cell, but common bacterial plasmids generally have many copies per cell and will continue to be copied and replicated with the cells (unless there is selective pressure to loose the plasmid).
On Tue, Sep 19, 2017 at 11:49 AM Rikke Rasmussen <rikke.c....@gmail.com> wrote:
In bacteria, at least, the plasmids are replicated with the cell cycle - it's how "we" make more plasmid. =)
On Tue, Sep 19, 2017 at 9:33 AM, Cory Geesaman <co...@geesaman.com> wrote:
The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
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Cory J. Geesaman
Sep 19, 2017, 2:32:51 PM9/19/17
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What about within things like algae?
On Tuesday, September 19, 2017 at 12:49:55 PM UTC-4, Rikke wrote:
In bacteria, at least, the plasmids are replicated with the cell cycle - it's how "we" make more plasmid. =)
On Tue, Sep 19, 2017 at 9:33 AM, Cory Geesaman <co...@geesaman.com> wrote:
The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
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Skyler Gordon
Sep 19, 2017, 2:49:05 PM9/19/17
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There will always be a natural selection against the plasmid that may cause eventual deletion. Single cell organisms base their foundation on keeping everything very simple so adding a large amount of unnecessary DNA will take more energy to synthesize and maintain than just the needed chromosomal DNA. That being said, there are strains of bacteria (E.coli DH5-alpha is a common one) that are engineered to preserve plasmids over a long period of time.
Typically with a system like algae, plasmids will be constructed and preserved in an E.coli host strain and then moved over to the desired algae strain. I don't know how well an algae strain will preserve a plasmid over time, but if that is a concern (you need to maintain the culture at room temperature or above for a long period of time and the plasmid does not contain information necessary for survival) inserting directly to the genome with a CRISPR technique could be possible. You may be able to skip the plasmid insertion all together by conjugating CRISPR with a TEF2 like protein, but I would have to do some research to really say how.
-SG
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Cory J. Geesaman
Sep 20, 2017, 2:06:07 AM9/20/17
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The CRISPR+TEF2 piece is interesting - I was looking at electroporation as a relatively simple way to get the DNA into a cell which is why I was thinking of plasmids.
Ravasz
Sep 23, 2017, 10:10:00 AM9/23/17
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Hi,
I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.
As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.
Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.
For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.
If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.
Cheers,
Mate
I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.
As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.
Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.
For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.
If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.
Cheers,
Mate
Mac Davis
Sep 23, 2017, 1:59:32 PM9/23/17
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Why does nobody use plasmids to stably create proteins in eukaryotic cells?
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Skyler Gordon
Sep 23, 2017, 5:54:38 PM9/23/17
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Beware: I've been told that "pure" cultures of specific algae are difficult to achieve and maintain.
-SG
-SG
On Sat, Sep 23, 2017 at 10:59 AM Mac Davis <mac.t...@gmail.com> wrote:
Why does nobody use plasmids to stably create proteins in eukaryotic cells?
On Sep 23, 2017 9:10 AM, "Ravasz" <ravasz...@gmail.com> wrote:
Hi,--
I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.
As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.
Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.
For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.
If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.
Cheers,
Mate
On Tuesday, 19 September 2017 17:33:09 UTC+1, Cory J. Geesaman wrote:The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
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Máté Ravasz
Sep 25, 2017, 8:50:50 AM9/25/17
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"Why does nobody use plasmids to stably create proteins in eukaryotic cells?"
Because unlike bacteria, eukaryotic cells have chromosomes and they can't handle plasmids too well. When an eukaryotic cell replicates it will first duplicate its genome using multiple replication origins on each chromosome and then segregate the new chromosomes into daughter cells evenly. Special care is taken that one and only one copy of each chromosome is distributed to each daughter cell. This is regulated by the centromere of the chromosome. Plasmids lack both of these structures: they do not have the multiple replication origins that chromosomes do, and they do not have a centromere. So they are not reliably replicated, and then they are not evenly distributed to the daughter cells, so after a few rounds of replication they will be lost.
Also plasmids only work for up to about 15kb length, bigger plasmids will not be maintained in bacteria where you need to prepare them before inserting them into eukaryotes. Unfortunately, both the centromeres and eukaryotic replication origins tend to be several megabases in length, so you cannot put them into plasmids as they are just too big. So plasmids are too small to be able to contain the appropriate machinery to stably express protein in eukaryotic cells.
Also plasmids only work for up to about 15kb length, bigger plasmids will not be maintained in bacteria where you need to prepare them before inserting them into eukaryotes. Unfortunately, both the centromeres and eukaryotic replication origins tend to be several megabases in length, so you cannot put them into plasmids as they are just too big. So plasmids are too small to be able to contain the appropriate machinery to stably express protein in eukaryotic cells.
Therefore in eukaryotes other strategies are used: artificial chromosomes which are much larger than a plasmid, integrating plasmids which fuse into a host chromosome, or specially engineered plasmids which borrow mechanics from viruses to overcome these limitations.
Mate
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Cory J. Geesaman
Oct 1, 2017, 12:59:14 AM10/1/17
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Is it possible to create a synthetic chromosome containing a compilation of all the plasmids you want for eukaryotic cells to get them to replicate?
On Monday, September 25, 2017 at 8:50:50 AM UTC-4, Ravasz wrote:
"Why does nobody use plasmids to stably create proteins in eukaryotic cells?"Because unlike bacteria, eukaryotic cells have chromosomes and they can't handle plasmids too well. When an eukaryotic cell replicates it will first duplicate its genome using multiple replication origins on each chromosome and then segregate the new chromosomes into daughter cells evenly. Special care is taken that one and only one copy of each chromosome is distributed to each daughter cell. This is regulated by the centromere of the chromosome. Plasmids lack both of these structures: they do not have the multiple replication origins that chromosomes do, and they do not have a centromere. So they are not reliably replicated, and then they are not evenly distributed to the daughter cells, so after a few rounds of replication they will be lost.
Also plasmids only work for up to about 15kb length, bigger plasmids will not be maintained in bacteria where you need to prepare them before inserting them into eukaryotes. Unfortunately, both the centromeres and eukaryotic replication origins tend to be several megabases in length, so you cannot put them into plasmids as they are just too big. So plasmids are too small to be able to contain the appropriate machinery to stably express protein in eukaryotic cells.Therefore in eukaryotes other strategies are used: artificial chromosomes which are much larger than a plasmid, integrating plasmids which fuse into a host chromosome, or specially engineered plasmids which borrow mechanics from viruses to overcome these limitations.Mate
On 23 September 2017 at 18:59, Mac Davis <mac.t...@gmail.com> wrote:
Why does nobody use plasmids to stably create proteins in eukaryotic cells?
On Sep 23, 2017 9:10 AM, "Ravasz" <ravasz...@gmail.com> wrote:
Hi,--
I'm a bit late to the party but I wanted to chime in as I did my PhD on designing self-replicating plasmids for humans and now I am trying to engineer algae as a hobby.
As the previous replies already correctly stated, there is a mayor difference between prokaryotes and eukaryotes. Algae is a blanket term that encompasses organisms from both these domains so depending on exactly what species you want to work with you will need to go on very different routes.
Bacteria, as said by others here before me, will usually take up any plasmid that has a reasonable size and a matching replication origin. Most bugs will then gradually lose whatever genes you put on that plasmid as they will only want to hang on the selection markers which are hopefully encoded on your plasmid, while they will gradually switch off any other genes that don't provide them a growth advantage. You need specifically engineered bacteria that do not do that and keep expressing your genes of interest, but I'm afraid most popular alga species are notorious for having to be retransformed regularly as they just keep losing expression of any product they should be making.
For eukaryotes getting a plasmid to replicate in them is more tricky than it should be. It can be done but replication origins tend to be huge, complex and require several genes to work. I am happy to go into detail if interested, but by and large no one uses plasmids to stably express proteins in eukaryotes. Instead its a lot more popular to integrate the plasmid into the host genome, and this instantly solves the whole hassle of having to maintain an extra plasmid. So if eukaryotic algae are your thing, then you might want to use integrating plasmids for stable expression of any product you might need. Crispr-Cas9 or TALENs are also an option but these are more useful if you want to modify already existing genes rather than putting in something completely new.
If you feel like giving a bit more detail on what species you plan on using and what you want to achieve with it, then I think we can give you more specific information.
Cheers,
Mate
On Tuesday, 19 September 2017 17:33:09 UTC+1, Cory J. Geesaman wrote:The title pretty much has the substance of this. I'm curious if you can introduce plasmids to a cell via electroporation and have those plasmids replicated in subsequent cell divisions, or if they only end up in one of the cells while multi-generational versions must be incorporated into the DNA somehow. Would the answer differ for different types of cells?
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Skyler Gordon
Oct 1, 2017, 1:19:26 AM10/1/17
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You're going to need either a really good polymerase or a lot of ligase.
-SG
-SG
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Nathan McCorkle
Oct 2, 2017, 2:34:07 PM10/2/17
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On Sat, Sep 30, 2017 at 9:59 PM, Cory J. Geesaman <co...@geesaman.com> wrote:
> Is it possible to create a synthetic chromosome containing a compilation of
> all the plasmids you want for eukaryotic cells to get them to replicate?
Sure, check out YACs (Yeast Artificial Chromosomes)
> Is it possible to create a synthetic chromosome containing a compilation of
> all the plasmids you want for eukaryotic cells to get them to replicate?
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