In bacteria, DNA is found in a large, primary chromosome, but many also harbor smaller, circular pieces of DNA called plasmids. These extrachromosomal elements replicate independently of the main chromosome. A key characteristic of a plasmid is its “copy number,” which refers to the average number of copies found within a single bacterial cell. This number is a regulated feature that can range from a handful to several hundred copies, depending on the specific plasmid.
The Significance of Copy Number
The number of plasmid copies in a cell directly influences gene expression. Genes on a plasmid are templates for producing proteins, and more templates lead to higher production. This makes high-copy-number plasmids, which can exist in 500-700 copies per cell, useful tools for manufacturing large quantities of a specific protein. For instance, using a plasmid that replicates prolifically ensures a high yield of a therapeutic enzyme from the bacterial culture.
This high production capacity creates a “metabolic burden” on the host cell. Maintaining hundreds of plasmids and constantly expressing their genes requires significant energy and cellular resources. This energetic drain can slow the cell’s growth, affect its health, and sometimes lead to aggregated, non-functional proteins. If the protein being produced is toxic, a high copy number could kill the host cell.
Consequently, low-copy-number plasmids, maintained at a stable 1-10 copies per cell, offer a more balanced solution. They impose a much smaller metabolic load, making them ideal when cell stability and long-term expression are more important than sheer volume. This is useful when expressing proteins that are somewhat toxic or when engineering complex metabolic pathways where overloading the cell could disrupt the system.
Biological Control of Replication
The primary determinant of a plasmid’s copy number is a specific DNA sequence on the plasmid itself known as the origin of replication (ori). This region acts as the starting point for DNA replication and contains the elements that regulate how often the plasmid is copied. The host cell’s replication machinery is recruited to the ori, but the control over the frequency of this event lies with the plasmid’s regulatory system.
Replication control mechanisms are categorized as either stringent or relaxed. Stringent control is characteristic of low-copy-number plasmids, present in as few as one to five copies per cell. This regulation involves plasmid-encoded proteins that bind to repeated DNA sequences, called iterons, within the ori to limit replication initiation. Such systems ensure the plasmids are duplicated reliably but not excessively.
Relaxed control, in contrast, allows for a much higher copy number, with some plasmids reaching 15 to over 700 copies per cell. A well-studied example is found in the ColE1 family of plasmids. This system uses two small RNA molecules, RNA I and RNA II, encoded by the plasmid. RNA II acts as a primer to start DNA replication, but as plasmid numbers increase, so does the inhibitory RNA I, which binds to RNA II and prevents further replication in a negative feedback loop.
External Factors Influencing Copy Number
While the origin of replication sets a plasmid’s baseline copy number, it is not absolute and can be influenced by external factors. The genetic background of the host bacterial strain plays a part, as some strains are better equipped to handle the metabolic demands of numerous plasmids. Strains like DH5α and TOP10 are used in laboratories because they lack certain enzymes that can degrade plasmid DNA, facilitating the maintenance of high-copy plasmids.
Growth conditions also have a measurable impact on the final plasmid count. Temperature is a key variable; for some plasmids, a lower growth temperature can reduce metabolic stress and support more stable plasmid maintenance. The composition of the growth medium, including nutrient availability, affects the overall health and growth rate of the host cells, which in turn influences their capacity for plasmid replication.
The physical characteristics of the plasmid itself can also modify its copy number. Larger plasmids are more difficult for the cell’s machinery to replicate and tend to be maintained at lower copy numbers than smaller plasmids, even if they share the same origin of replication. Adding a large or complex gene insert to a high-copy plasmid can also lead to a lower-than-expected yield.
Practical Applications in Research and Industry
Scientists select plasmids with specific copy numbers to optimize their experimental or industrial processes. For large-scale manufacturing of biopharmaceuticals, such as producing human insulin in E. coli, high-copy-number plasmids are used. Using a vector that generates hundreds of copies per cell maximizes the gene dosage, leading to the high protein yields required for therapeutic use.
In metabolic engineering, where the objective is to build or modify complex biochemical pathways, low-copy-number plasmids are frequently preferred. Researchers may need to express multiple enzymes in a balanced ratio. Using low-copy vectors prevents the overproduction of any single enzyme, which could create a metabolic bottleneck or be toxic to the cell, ensuring the engineered pathway functions smoothly.
Researchers do not just assume a plasmid’s copy number; they actively verify it as part of their workflow. A common technique for this is quantitative PCR (qPCR), a method that can accurately measure the number of plasmid DNA molecules relative to the number of host cell chromosomes. This verification step ensures the experimental system is behaving as expected and allows for reliable optimization of protocols.