How and Why Does Plasmid Copy Number Change?

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18
Nov
Plazmid Kopya Sayisi Nasil ve Neden Degisir
Date November 18, 2022
Categories Newsletter

How and Why Does Plasmid Copy Number Change?

We have talked about what plasmids are and their applications in previous newsletters. Let’s take a closer look at plasmid copy numbers and the ways they can be useful in the lab.

The contents of this month’s newsletter;

  • What exactly does plasmid copy number mean?
  • Why is plasmid copy number important?
  • It will be on how the plasmid copy number can be changed.

Plasmid copy number can have a big impact on yield, and while sometimes it is best to have a high plasmid DNA copy number, other times it can be a disadvantage.

What is Plasmid Copy Number?

Plasmid copy number refers to the number of molecules of a single plasmid present in the host bacterial cell. They are expressed as low (15-20 copies per cell), medium (20-100 copies per cell) or high (500-700 copies per cell) copy numbers and vary in size depending on three main factors.

1) Ori and its components (e.g. ColE1 RNA I and RNA II).

2) The size of the plasmid and its associated attachment (larger attachments and plasmids can be replicated in a lower number as they represent an enormous metabolic burden for the cell).

3) Culture conditions (factors affecting the metabolic load on the host).

Why Knowing the Copy Number of Your Plasmid is Important

It is very important to know which category your plasmid falls into before you start working. If you know that you are working with a low copy number plasmid, you should not be too surprised by the low yield and you can decide to create more culture media.

One advantage of a high plasmid copy number is the higher stability of the plasmid. But this is not to say that the opposite does not happen!

When is a High Plasmid Copy Number Important?
– If you are experiencing low protein yields when performing protein production studies, a high copy number of plasmids, possibly due to a high metabolic load, can lead to protein aggregation and insufficient modification.
– Using high plasmid copy numbers in cloning studies will result in higher yields.
When is Low Plasmid Copy Number Good?
– While a high copy number is always considered good, some situations are better suited to a low copy number. For example, if you want to study a fungal protein for its antibacterial properties, it might be good to work with a low plasmid copy number to minimize toxic effects and not kill the bacteria.
– Overexpressed proteins, incorrect protein-protein pairings can lead to structural problems in the protein itself.
How do we change the plasmid copy number?

For the reasons mentioned above, it can be advantageous to have different plasmids with different copy numbers to choose from when doing your studies. Detailed studies on plasmid replication have paved the way for how we should vary plasmid copy number:

– You can increase the copy number of some plasmids by growing the host bacteria at high temperatures.

– Exposure of the host bacterium to chloramphenicol inhibits bacterial protein synthesis, leading to chromosomal replication inhibition and cell division inhibition. Plasmids require long-lived proteins and continue to replicate even when replication and cell division have stopped. Eventually, when the environment runs out of proteins, plasmid replication will stop, but the average copy number will have increased significantly.
Author Letgen Biotech