Did you ever get caught passing a note in class? Or the more modern-day equivalent, were you ever caught sending a text message in class?

Imagine, for a moment, if that note came with the power of putting a force field around you so that the teacher couldn’t confiscate the note and read it out loud in front of the entire class.

In nature, plasmids are kind of like a note that is passed between different bacterial cells that also gives them superpowers such as resistance to antibiotics. Plasmids and their superpowers are workhorse tools in molecular biology that enable a wide range of powerful experiments.

A plasmid is a small, circular, double-stranded DNA molecule that is distinct from a cell’s chromosomal DNA. Plasmids are naturally found in bacteria, and they are also important scientific tools used in molecular biology for gene cloning, protein expression, luciferase reporter assays, genetic and RNA interference screens, and more.

In this article we’ll discuss the basics of DNA plasmids and explain why they are such versatile tools.

 

In this article:

Naturally occurring plasmids

Key features of a plasmid

Gene of interest

Promoter

Multiple cloning site

Selection marker

Plasmid uses

Molecular cloning

Protein expression

Luciferase reporter assays

Genetic and RNA interference screens

References

 

Naturally occurring plasmids

Plasmids, also referred to as vectors or constructs, are a means for sharing genetic information with other cells in a process called horizontal gene transfer (HGT). Why do bacteria transfer genes to other cells? Well, there’s a bunch of reasons for this kind of transfer.

You can think about the world of microorganisms like a battlefield; bacteria are constantly fending off attackers or going on the attack themselves.

A plasmid is a versatile tool for bacteria in this molecular warfare. It defends bacteria from the antibiotics of other attacking cells. Plasmids also enable bacteria to attack different types of bacteria and protect their clonal siblings from friendly fire.

Plasmids also have many other roles such as helping bacteria adapt to harsh environments, causing outbreaks of food poisoning, and even sending love notes to other bacteria instructing them to form a sex pilus between two cells for direct genetic transfer (Addgene, 2024).

Because plasmids have the ability to transfer genetic information to other bacteria, plasmids are an important functional tool used by scientists for a variety of experiments. Before we get to some of those experiments let’s discuss the basics of what makes a plasmid, a plasmid.

 

Key features of a plasmid

Scientists also use plasmids as research tools to generate specific biomolecules and carry out a variety of experiments.

There are a few key features of plasmids in molecular biology (Figure 1):

·         Gene of interest

·         Promoter

·         Selection marker

·         Origin of replication

·         Multiple cloning site

We’ll cover a few of these key features here, but if you’re interested in learning more about any of these then this article is a great reference.

Figure 1. Key features of a plasmid include the gene of interest (orange), promoter (grey), selection marker (green), and origin of replication (magenta).

 

Gene of interest

The first key feature is the gene of interest. In the context of cloning or protein expression, this is the gene that encodes the protein that you’re interested in studying. For luciferase reporter assays, the gene of interest is luciferase, whose expression is under the control of a promoter that is responsive to a transcription factor or signaling pathway. And for genetic screens, the gene of interest will be genes coding for different kinds of RNA used in your genetic or RNA interference screen.

We’ll discuss more about each of these genes of interest in their respective sections below.

 

Promoter

The promoter is the stretch of DNA where proteins first bind to before they transcribe the gene of interest into RNA.

In most plasmids, the promoter is like a supporting actor; important, required even, but outshone by the main actor which is the gene of interest.

For example, T7 is a common promoter for bacterial plasmids because many competent cells such as BL21(DE3) and DL39(DE3) are engineered to express T7 polymerase upon IPTG induction (Figure 4). In fact, that’s what the (DE3) in their name stands for.

For luciferase reporter plasmids, however, the promoter gets to be the star of the show! In this case, the supporting gene of interest is always luciferase. But the promoter will be different depending on what kind of luciferase reporter you’re working with. Different promoter DNA sequences will bind to distinct transcription factors and “report” on their activity.

We’ll discuss promoters more in the section on luciferase reporter plasmids.

 

Multiple cloning site

The multiple cloning site surrounds the gene of interest and is used to insert a new gene of interest into a plasmid, or to take an old gene of interest out of a plasmid. It consists of DNA sequences that are recognized and cut by molecular scissors called restriction enzymes (Figure 2).

Just like the transcription factors we just discussed, there are a lot of different restriction enzymes that each bind to and cut distinct DNA sequences.

This gives you options in terms of what enzymes to use, which is good because with traditional cloning techniques it’s important that the restriction enzyme DNA recognition sequence does not also exist within your gene of interest. Otherwise, when you go to prepare the gene of interest to put it in your plasmid, you will also cut within that gene.

Figure 2. The multiple cloning site is DNA sequences (lime green) that flank the gene of interest (orange). Restriction enzymes (purple scissors) cut at restriction sites on the multiple cloning site allowing the insertion and removal of different genes from the plasmid.

 

However, multiple cloning sites are quickly becoming a relic of traditional cloning techniques so you may not need a multiple cloning site depending on how exactly you do your cloning. We’ll discuss this more in the section about molecular cloning.

 

Selection marker

Another key feature worth talking about here is the selection marker, which helps select for cells that contain the plasmid. For example, the selection marker often is a gene that expresses a protein that makes the cell resistant to antibiotic treatment. So when you treat your cells with antibiotics, the cells with the plasmid inside will keep growing whereas cells without the plasmid will die or have their growth stall.

Why this is important is because when you grow your cells on plates, without some sort of selectable marker, you have no way of telling which colonies have cells that took up your plasmid that holds your gene of interest and which ones do not.

By using an antibiotic resistance gene as a selection marker, when you plate cells on media that has the given antibiotic, only cells carrying your plasmid with the resistance gene and gene of interest will live.

We have a great video that goes into more detail about the concept of selection and dropout media. 

 

Plasmid uses

Now that we’ve covered the important parts of a plasmid, let’s discuss some of the techniques and experiments that use plasmids.

 

Molecular cloning

If you want to make more of the same plasmid, that is easy. You just transform competent cells that are compatible with your plasmid, like GB5-alpha^TM for example, grow the cells up, and then harvest the plasmid DNA.

When we talk about molecular cloning, we’re really discussing how to make a new plasmid that doesn’t exist or that you don’t have access to.

For example, let’s say you have “Gene X” in your pUC19 cloning plasmid, but you want to have “Gene Y” in there instead. Nowadays there are many ways to clone, but we’ll cover the traditional restriction-enzyme based method for cloning.

First, you would choose restriction enzymes with recognition sequences in the multiple cloning sites and use those to cut out the old Gene X. You can separate your cut plasmid and Gene X by running this sample on an agarose gel and cutting out the band for the plasmid. 

Second, insert the new Gene Y with overlapping DNA sequences. Usually this requires a PCR (polymerase chain reaction) step to generate the insert. Fuse together the plasmid and Gene Y using DNA ligase.

Third, transform the plasmid with Gene Y into GB5-alpha^TM or another type of compatible competent cells to scale up your new plasmid (Figure 3).

Figure 3. To clone a new gene into a plasmid you first (left) cut out the old gene (orange) with restriction enzymes (purple scissors), then (middle) insert the new gene (blue) into the plasmid, and lastly (right) transform the new plasmid into competent cells.

 

You want to make sure that you cloned your new plasmid correctly. We cover three common ways to check your plasmid in this article.

DNA cloning is an essential step in bioscience research. Cloning the right plasmid(s) is a prerequisite for each of the following examples, unless you already have your plasmid ready to go.

 

Protein expression

Molecular cloning is a foundational technique in life sciences research, and in many ways purifying and using recombinant proteins is right behind cloning in terms of importance and prevalence.

The first step in generating a purified recombinant protein is to express the protein in a host organism. Escherichia coli (E. coli) expression strains, such as BL21, are the most common choice for protein expression.

After you’ve cloned your protein expression plasmid in the previous step, you’re ready to transform the cells with your plasmid, then induce protein expression with IPTG (Figure 4).

 

Figure 4. IPTG induction turns on the expression of T7 RNA polymerase (purple) which transcribes the gene of interest (orange) in the plasmid.

 

After expressing your protein, there are a few different ways to purify the protein, which we cover in this article. Check it out if you’re interested in learning more about protein purification

Luciferase reporter assays

Luciferase reporter assays are tools often used in the gene regulation field. In these plasmids, luciferase is the gene of interest, and different promoters will be used depending on which signaling pathways and downstream transcription factor you’re studying.

Signaling pathways are the way that cells communicate with their surrounding environment. An external signal, such as low oxygen, is transmitted through a series of different proteins. You can think about this like the telephone game where each person whispers what they heard from the previous person, on down the chain. Fortunately for us, signaling pathways are usually much more reliable in transmitting the correct signal as compared to the telephone game.

Ultimately, this signal ends with activating transcription factors, which then initiate gene transcription programs to help cells respond to the environmental stimulus that kicked off the signaling pathway in the first place. Transcription factors do this by binding to particular DNA “recognition” sequences and recruiting other proteins that then transcribe those genes into RNA. Usually that RNA is processed into mRNA and translated into proteins that help the cell respond to the initial stimulus, such as low oxygen.

In the context of a luciferase reporter plasmid, however, the higher transcription from the specific promoter will lead to more luciferase accumulating in a cell which will increase the bioluminescent signal from that sample (Figure 5). The signal from luciferase oxidizing luciferin is the way that the plasmid “reports” on the activity of the signaling pathway or transcription factor.

Figure 5. A luciferase reporter assay for HIF1 (left) is transfected into mammalian cells where luciferase will be expressed according to the activity of the HIF1 transcription factor in that cell. The more active HIF1 is, the more luciferase is produced resulting in higher bioluminescent signal.

 

See Table 1 for the list of GoldBio’s luciferase reporter plasmids, including the signaling pathway and transcription factors that those plasmids report on.

 

Table 1. GoldBio’s luciferase reporter plasmids.

Plasmid	Signaling pathway	Transcription Factor
pGL3-BRE-Luciferase	Bone Morphogenetic Protein (BMP)	SMAD (Small worm phenotype and Mothers against decapentaplegic)
4xCSL-Luciferase	Notch	CSL (CBF1/Suppressor of Hairless/LAG-1)
HRE-Luciferase	Hypoxia	HIF1 (Hypoxia inducible factor 1)
3xERRE-Luciferase	Estrogen	ER (Estrogen receptor)
TFEB-promoter-Luciferase	mTOR	TFEB (Transcription factor E-box binding)
pGL3-RARE-Luciferase	Retinoic acid	RAR (Retinoic acid receptor)
MicroRNA Studies Luciferase	Depends on miRNA	miRNA

 

Genetic and RNA interference screens

Genetic and RNA interference (RNAi) screens are powerful tools for discovering new biology. For example, if you wanted to find the genes that enable HIV replication, analyze the signaling pathways we discussed in the last section, or identify important genes for a particular cancer, then these screens can help you discover that information.

In the previous examples in this article, we talked about experiments that use a single plasmid. Genetic and RNAi screens are at another scale; you’ll want to make at least tens of thousands of plasmids for these experiments. The gene of interest for each plasmid will encode for a guide RNA (gRNA) for a CRISPR screen, or a short (shRNA), silencing (siRNA), or micro (miRNA) RNA for an RNAi screen (Figure 6).

Figure 6. Plasmids with genes encoding guide RNAs (gRNA) are used for CRISPR screens, and plasmids with genes encoding micro RNAs (miRNA), silencing RNAs (siRNA), and short hairpin RNAs (shRNA) are used for RNA interference screens.

 

Each plasmid will have a different gene of interest, which will encode one gRNA, shRNA, siRNA, or miRNA that will target a cellular gene to either silence it (greatly reduce its expression: shRNA, siRNA, miRNA) or delete the gene (eliminate its expression: gRNA).

Selecting the plasmids with the desired impact is usually done by sequencing and counting how many of each type of plasmid is present at the end of the screen. In a positive selection screen, there will be more of the plasmids that have the desired impact. In contrast, in a negative selection screen, there will be less of the plasmids that have the desired impact (Figure 7).

Figure 7. From a starting pool of different plasmids (left), a screen with positive selection will enrich for the plasmid(s) of interest (top right), whereas a screen with negative selection will deplete the plasmid(s) of interest (bottom right). 

 

So that’s our high-level overview of plasmids. We have a lot of great resources on related topics that we’ve included links to throughout the article and below. Follow those links if you’re interested in learning more about plasmids and the experiments they enable. Additionally, GoldBio sells many plasmids and related reagents for the experiments that we discussed including competent cells, antibiotics, IPTG, and more. Click on the links in this article or go directly to our website so that our reliable and affordable products can accelerate all of your plasmid-related research!

 

 

References

Addgene. (2024, October 8). Plasmids 101: The wide world of natural plasmids. Addgene Blog. https://blog.addgene.org/plasmids-101-natural-plasmids

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