Re: [DIYbio] Re: Do plasmids introduced via electroporation replicate with subsequent cell divisions?

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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