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How (and Why) To Manipulate Plasmid Copy Number

An abstract colourful picture of many numbers to depict the different plasmid copy numbers

Plasmid copy number can have an enormous impact on your yields, and sometimes having a high plasmid copy number is best, but other times it can be detrimental.

Join us as we take a closer look at plasmid copy number and examine ways this can be manipulated in the lab giving you flexibility in your work.

On the agenda:

  1. What exactly does plasmid copy number mean?
  2. Why is plasmid copy number important?
  3. How can plasmid copy number be manipulated?

What is Meant by Copy Number?

Plasmid copy number refers to the average or expected number of copies per host cell. Plasmids are either low, medium, or high copy number. Plasmids vary widely in copy number depending on three main factors:

1) The ori and its constituents (e.g., ColE1 RNA I and RNA II).

2) The size of the plasmid and its associated insert (bigger inserts and plasmids may be replicated at a lower number as they represent a tremendous metabolic burden for the cell).

3) Culture conditions (i.e., factors that influence the metabolic burden on the host).

QIAGEN has a helpful table with a selection of currently available plasmids and their respective copy numbers. While there are differences in the way plasmids are categorized according to copy number, a very general rule of thumb is shown in Table 1.

Table 1. Average copy numbers for low, medium, and high copy number plasmids.

Category

Typical number of copies per bacterial cell

Low copy (e.g. pBR322 and derivatives)

15–20 copies per cell

Medium copy

20–100 copies per cell

High copy (e.g. pUC18, pUC19 vectors)

500–700 copies per cell

Why is it Important to Know the Copy Number For Your Plasmid?

It’s crucial to know which category your plasmid falls into before starting your experiment. If you know you are working with a low-copy number plasmid, you shouldn’t be too surprised with a low yield, and you might decide to set up more cultures.

On the other hand, if you get a poor yield from a high copy plasmid, you know you need to do some troubleshooting, assuming your insert is not too large!

An advantage of a high plasmid copy number is the greater stability of the plasmid when random partitioning (i.e., partitioning of plasmids into daughter cells) occurs at cell division. However, a high number of plasmids can also result in lower yields.

Let’s look at a few cases where you really need to consider copy number in your experiments:

When is a High-Copy Number Good?

There are several situations where you want a high copy number plasmid, including:

  • Protein expression
    Although there is no significant advantage to using higher-copy-number plasmids over pBR322-based vectors in terms of protein production yields, a high-copy plasmid might be your first port of call if you experience low protein yields.

    Bear in mind that a very high-copy number can lead to protein aggregation and deficient post-translational modification, presumably because the metabolic burden is too high.
  • Cloning
    Using a high-copy plasmid will generally result in greater yields from plasmid preps.

When is a Low Copy Number Good?

You might think that a high copy number is always best, but some situations are better suited to a low copy number.

  • Expressing a toxic product
    Let’s say you want to study a fungal protein for its antibacterial properties, and you want to express it in bacteria. A low-copy number plasmid might be better to minimize toxic effects and avoid killing your bacterial cultures.
  • Mutant Studies
    You have mutated your enzyme of interest. Now you want to compare its activity to the wild-type enzyme in a physiological context (i.e., transform it into native host cells).

    Low-level expression from a single copy is usually a better option to increase the chances of physiologically relevant measurements and to assess in vivo phenotypes.

    Over-expressed proteins may generate artificial phenotypes, false protein-protein interactions, and structural issues within the protein itself, leading to confusing and unreliable results. (We all know that results can be confusing enough without further complicating things!)

How Can We Manipulate Plasmid Copy Number?

For the reasons given above, it can be advantageous to have a selection of plasmids with different copy numbers to choose from as you carry out your research. Much effort has gone into understanding how plasmid replication is controlled, paving the way for us to manipulate this plasmid copy number. Let’s look at a couple of options available to us.

Induced Amplification by Temperature Shift and Altering Bacterial Growth Rate

You can increase the copy number for some plasmids by growing the host at elevated temperatures. This could be the case for pBR322 because the bacterial growth rate influences the fine-tuning of the RNA I/RNA II regulation. [1] 

Chloramphenicol Amplification

Chloramphenicol amplification works for many low-copy plasmids containing the pMB1 origin [2]. The main steps are:

  • Exposure of the host bacterial culture to the antibiotic chloramphenicol inhibits bacterial protein synthesis.
  • This leads to chromosomal replication inhibition because this also relies on ongoing protein synthesis and inhibition of cell division.
  • Plasmids only require more long-lived proteins and continue to replicate even though chromosomal replication and cell division have stopped.
  • Eventually, plasmid replication stops when the cell becomes exhausted (proteins used up), but the average copy number will have increased significantly.

Unintentional Manipulation of Plasmid Copy Number

As mentioned above, the plasmid insert can also influence copy number. For example, a high-copy pUC plasmid may replicate at medium or low copy numbers when ligated to very large DNA inserts, resulting in lower plasmid yields than expected.

This decreased yield is because plasmid replication is a metabolic burden for the host cell. If the burden becomes too large (e.g., massive inserts, elevated growth temperature), plasmid-bearing cells will become less efficient, and growth will slow down.

Eventually, the culture will become dominated by existing plasmid-free cells, leading to low plasmid yield.

Plasmid Copy Number Summarized

Plasmid copy number can influence experiments, and while it is appealing to have high-copy numbers for your plasmids, there are some applications where low plasmid copy numbers are best. You can achieve the desired copy number by using an appropriate plasmid or copy number manipulation.

Do you know of any other methods to increase the plasmid copy number? If you have some ideas not mentioned here, we would love to hear from you!

Don’t forget to check out the related articles on the origins of replication and the high-copy pUC18 plasmid.

This article was originally published on January 14, 2015. Reviewed and updated May, 2022.

References:

  1. Lin-Chao S, Bremer H. 1986. Effect of the bacterial growth rate on replication control of plasmid pBR322 in Escherichia coli. Mol Gen Genet. 203(1):143–9.
  2. Howe C. Vectors, transformations and hosts. In Gene Cloning 2nd Ed. Cambridge University Press, 2007. ISBN: 9780521521055

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5 Comments

  1. Alex on August 9, 2016 at 7:48 am

    Would the chloramphenicol application work also for low copy plasmids other than those with pMB1 origin?



  2. JDMoore on February 25, 2016 at 9:42 am

    Great stuff, Karen. Can you comment on the use of plasmids with high v. medium v. low copy numbers in studies that are NOT studying the plasmid, but are instead solely using the antibiotic resistance gene-containing plasmid to lower the possibility of contamination?? It sounds like you would expect that high copy number, e.g. pUC-derived, plasmids could interfere with unrelated cell processes, e.g., metal metabolism???

    Thanks!



    • Karen OHanlon Cohrt on June 10, 2016 at 7:52 am

      Hi JD,

      Apologies for late reply. For some reason, I don’t get notifications when someone comments on my article!

      If I understand your question correctly, and you only want to use plasmids to select for organisms which contain them in order to avoid contamination, then I don’t think it matters too much whether your plasmid is low, medium or high copy number. Have you found it does make a difference?

      Best wishes,
      Karen



  3. Karen O'Hanlon Cohrt on February 14, 2015 at 3:27 pm

    Hi Michael,

    You are right. There are a few typos in the article concerning the naming of pBR322. Thanks for pointing that out. I will ask our admin to fix it.

    Best Regards,
    Karen



  4. MichaelAdamat on February 10, 2015 at 9:49 am

    In the article you are writing about pBR22 (not pBR322) a number of times. Since the paper you refer to is about pBR322, it would probably be a good idea to check the spelling.



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