Have you heard of DNA plasmids, sometimes called DNA vectors, but you aren’t quite sure what exactly they are? If so, this is the perfect article for you.
DNA plasmids are a cornerstone of modern molecular biology. They make use of the gene as a fundamental biological unit and enable the careful and functional dissection of genes and their encoded proteins.
Plasmids are circular pieces of DNA normally found in bacteria and certain kinds of eukaryotes. In molecular biology, plasmids are used to express a gene and protein of interest in host cells or to provide cells with some sort of novel function or diagnostic feature.
In this article we’ll discuss 5 key features of DNA plasmids and explain why they are so important for their function.
In this article:
5 key features of DNA plasmids are the (Figure 1):
- Gene of interest
- Selection marker, such as antibiotic resistance
- Replication origin
- Promoter
- Multiple cloning site
Figure 1. Key features of a DNA plasmid (gray circle)
include the gene of interest (Gene X), selection marker (AmpR), origin of
replication (Ori), transcriptional promoter (Promoter), and multiple cloning
site (MCS). The promoter and multiple cloning site are right next to the gene
of interest and are relatively small compared to the other features so a “zoom
in” of these features is on the right.
Gene of Interest
The first key feature of a plasmid is that it can function as a vector, or vehicle that carries a gene of interest – the gene a researcher is trying to study. One might be using a plasmid to purify a protein that this gene of interest encodes, or to provide those cells with a novel feature or diagnostic tool. For example, luciferase plasmids are used to read out the transcriptional activity of cells that they are transformed into.
Selection Marker
The selection marker is another gene expressed from the plasmid in order to select for cells that have the plasmid inside. For example, with bacterial plasmids, the selection marker is an antibiotic resistance gene. Cells that have that plasmid will be resistant to the corresponding antibiotic and will grow just fine in cultures with the antibiotic. However, when plated on media with a certain antibiotic, cells that were not successfully transformed with this plasmid will not grow.
We have a great video that goes into more detail about the concept of selection and dropout media.
Ampicillin and Kanamycin are two commonly used antibiotics that you may have heard of. The “AmpR” gene in the plasmid in Figure 1 provides resistance to ampicillin.
By the way, if you have a plasmid you’re working with and you want to quickly look up its antibiotic resistance – use this handy tool!
For other organisms, the selection process can work a little bit differently. For example, in baker’s yeast (Saccharomyces cerevisiae), the selection marker produces an essential amino acid. In that way, only yeast cells that have the plasmid will grow in media that lacks that amino acid. So, while the details for selection markers can be different between distinct organisms, the overall principle of restricting growth to cells that contain the plasmid is the same.
Replication origin
Replication origins are special pieces of DNA that DNA polymerases recognize and bind to initiate DNA replication. DNA polymerases are enzymes that assemble a new, complementary strand of DNA from an existing template (Figure 2). Our DNA has lots of replication origins in them, as does the DNA of most species (Hu and Stillman, 2023).
Figure 2. DNA polymerase (green oval) makes new double stranded DNA by adding nucleotides to existing single-stranded DNA (purple). DNA polymerase first binds to replication origins (Figure 1) to initiate this process.
DNA plasmids also feature a DNA replication origin so that the host protein machinery will make more copies of the plasmid. This is important so that when cells replicate into new daughter cells, those cells will each contain copies of the plasmid.
Having more copies of the plasmid via DNA replication is also important for many of the applications that plasmids are used for, such as the luciferase assays referenced above or for making lots of recombinant protein.
Promoter
The promoter is the region of DNA just upstream of your gene of interest where the host RNA polymerase will bind and transcribe your gene into RNA (Figure 3). There are different promoters that are used that lead to different levels of transcription.
Figure 3. RNA polymerase (green oval) transcribes RNA (orange) from an existing DNA template (purple). RNA polymerase first binds to the promoter on DNA (Figure 1) to initiate RNA transcription.
So, for example, if you are working with a protein that is toxic at high expression levels, you would probably want to use a weak promoter (Vandierendonck et al, 2023). Conversely, if you need high levels of protein expression for your application, then you probably want to use a strong promoter.
Regardless of which type of promoter you’re using, this region is essential for gene expression so if you’re having any issues with your plasmid, check to make sure the promoter hasn’t accumulated any detrimental mutations.
Multiple Cloning Site
In traditional methods of molecular cloning, genes were added and removed to plasmids using restriction enzymes. These enzymes recognize and cut at specific short DNA sequences.
So, you could cut a previous gene out of a plasmid, purify the cut plasmid, then insert a new gene with complementary overlapping regions (Figure 4).
Figure 4. Gene X is cut out of the plasmid using
restriction enzymes (left), then separated from the cut plasmid, and then Gene
Y is inserted into the plasmid using DNA ligases (middle and right).
Downstream of the promoter is a multiple cloning site which contains multiple restriction enzyme cleavage options for inserting a gene of interest (Figure 1). These sequences are critically important for generating new plasmids containing your gene of interest.
Some more modern methods of molecular cloning do not make use of multiple cloning sites in plasmids (Bird et al, 2022). If you clone using one of these methods, such as Golden Gate Cloning for example, then you don’t need to worry about whether the multiple cloning sites are correct, or if they even exist in your plasmids.
Verifying Key Plasmid Features
For restriction enzyme sites, it is easy and quick enough to confirm that those sequences are correct by using restriction enzymes to cut your plasmid and check to see that it makes the correct number and sizes of DNA pieces.
For all other key features we’ve discussed, the easiest way to make sure that they are correct is to sequence the plasmid. In a related article we cover sequencing techniques and suggest that nanopore sequencing is the quickest, cheapest, and easiest way to get your entire plasmid sequenced – check it out for more details on why that is.
Now that you know the 5 key features of DNA plasmids you can use this knowledge to verify that your current plasmids are working correctly and to help design new plasmids for your research needs.
