So far in this series, we have looked at origins of replication, we’ve discussed how plasmid replication is regulated in the popular pBR22 plasmid, and we’ve seen how a disturbance of this regulatory mechanism has given rise to the high-copy pUC18 plasmid.

Are you ready for more plasmid talk??

If so, keep reading, as we will now take a closer look at copy number and examine ways in which it can be manipulated in the lab giving you flexibility in your work.

On the agenda:

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

1. What does copy number mean again?

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 great metabolic burden for the cell).

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

An overview of the plasmids currently available and their copy numbers can be found here. While there appears to be discrepancy in the way plasmids are categorized according to copy number, a very general rule of thumb is shown in the table below.

CategoryTypical number of copies per bacterial cell
Low copy (e.g. pBR22 and derivatives)15–20 copies per cell
Medium copy20–100 copies per cell
High copy (e.g. pUC18, pUC19 vectors)500–700 copies per cell

1.  Why is copy number important?

Although it sounds obvious, knowing which category your plasmid falls into is very important when starting out 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 therefore decide to set up more cultures. On the other hand, if you get a poor yield from a high copy plasmid, then you know you have some troubleshooting to do, assuming your insert is not too large to begin with!

An advantage of high 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 as mentioned above. Let’s look at a few cases where you really need to consider copy number in your experiments:

2. When is high-copy number good?

  • Protein expression: Although there appears to be no significant advantage of 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 do experience low protein yields. Bear in mind that 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 lower-copy number good?

  • 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 might be better to minimize toxic effects and to 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). To increase the chances of physiologically relevant measurements and/or to assess in vivo phenotypes, low-level expression from a single copy is usually a better option. 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 all by themselves without further complicating things!)

 3. How can we manipulate copy number?

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

Induced amplification by temperature shift and altering bacterial growth rate

Copy number can be increased for some plasmids by growing the host at elevated temperatures. This could be the case for pBR22 because the fine-tuning of the RNA I/RNA II regulation is influenced by the bacterial growth rate.1

Chloramphenicol amplification2

This works for many low-copy plasmids containing the pMB1 origin:

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

Unintentional manipulation

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 is because plasmid replication is a metabolic burden for the host cell, and if the burden becomes too large (e.g. huge inserts, elevated growth temperature), plasmid-bearing cells will become less efficient and growth will slow down. This results in the culture being taken over by any existing plasmid-free cells, eventually leading to low plasmid yield.

Now that you (hopefully!) know a lot more about copy number and factors that will influence it, you are no doubt dying to get back into the lab and do some cloning! Do you know of any other methods to increase plasmid copy number? If you have some ideas not mentioned here, we would love to hear from you!

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