Working in a molecular biology lab you may be expected to replicate plasmids.
But to do that you'll first need to understand the mechanisms behind it all.
What is it that drives plasma replication?
The answers lie in the origin of replication.
Every plasmid has an area known as the replicon. Here the mechanism of replication can begin.
In the replicon you'll find the origin of replication, as well as all its control elements.
The origin of replication, or ori, is the place where DNA replication begins.
The origin of replications in plasmids are generally different from the ones used
to create the host's chromosomal DNA. But like their chromosomal counterparts, they still rely
on the host cells machinery to make additional copies.
We'll explain why that's important to keep in mind a little later.
For now let's take a closer look at the makeup of these plasmid oris.
Ori sequences are generally rich in adenine and thymine. AT base pairs are held together
with just two hydrogen bonds,
as opposed to the three hydrogen bonds that keep guanine cytosine base pairs together.
This means that AT base pairs separate more readily than CG base pairs.
As the pairs break apart, it gives the replication machinery room to move in and get down to business, making copies.
There are a lot of origins of replications that we could look at,
found in viruses or eukaryotic cells. But let's focus on oris founded bacterial plasmids.
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These ori can be either relaxed, or stringent.
In both cases the host cell's replication machinery is used.
However relaxed ori are regulated only by DNA contained on the plasmid itself
They don't require the host cells initiation proteins.
These tend to be high copy plasmids.
On the other hand stringent ori require additional initiation proteins,
synthesized by the host cell's chromosome, to start replication.
They tend to be low copy plasmids.
The positive and negative regulation on ori creates a delicate balance that results in copy
number of the plasmid. It only takes small changes in the ori to affect copy number of the plasmid.
For example the pMB1 ori maintains about 20 copies per cell.
But with just two mutations in the origin, the pUC ori can produce as many as 700 copies per cell.
There are a few things to keep in mind as you're selecting plasmids for your experiment.
Especially if you're planning on using plasmids in the same bacterium.
You'll need to ensure your ori are compatible, meaning they won't get in the way of each other's replication.
Using plasmids with the same ori causes them to compete for the cell's machinery,
making it more difficult to maintain the plasmid's copy numbers.
As a result, each incompatible plasmid
thinks the cell has more copies than it actually does, resulting in a lower than desired copy number.
In the lab, you'll be able to control the copy number to fit your experiment's needs.
Many cloning vectors are high copy number plasmids, meaning that you'll be able to obtain more plasmids from fewer cells.
But sometimes this can be a disaster! If your gene of interest is toxic
a large number of them can poison and kill the cells.
[Blugene sighs] In these cases it's better to stick with low copy plasmids.
There are a whole host of conditions to consider
while choosing an ori, and how that will determine how much, or how little your plasmids replicate.
The type of bacteria could be a deciding factor. While most strains of E.coli can be used to
propagate plasmids, some are more hospitable than others.
For example and a E.coli are best for high yields of plasmids, because they don't contain the endonuclease that can degrade plasma DNA.
Lab conditions can also affect copy number.
Aeration, temperature, culture volume, antibiotic, and medium all have their own effect on plasmid replication.
Both the freshness of the cultures and incubation time also play a large role in plasmid replication.
Freshly streaked bacteria have higher copy numbers so pick a single colony,
and don't subculture from glycerol stocks, agar stabs, or liquid cultures to get your best yield.
If you really need to get the highest yield possible, start from a
fresh transformant colony and not just a fresh colony from streaking a frozen culture.
You'll always want to aim for 12 to 16 hours of incubation if you're looking for higher copy numbers .
In this window the bacteria just reached stationary phase, but the cells have not started to die off.
Now that you've got those tips and tricks it's time to get in the lab and replicate those plasmids.
We hope you enjoyed this video in our Plasmids 101 series. Don't forget to subscribe to
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