Everything You Need To Know About Plasmids, Vector, And Isolation

Plasmids are important for things like adapting to altering environments and bacterial evolution.

They carry genes with beneficial traits for bacteria to have, allowing them to continue to survive. Even in a single bacterial cell, many types of plasmids can happily coexist.

Everything You Need to Know About Plasmids, Vector, and Isolation

To geneticists and those in molecular biology, plasmids are incredibly important. In genetic engineering, plasmids play a crucial role in many procedures.

These procedures include recombinant protein production (such as human insulin), gene therapy research, and gene cloning. Without plasmids, none of these would be possible.

Generally, in order to do any of these procedures, a plasmid gets cut at a specific site, or various sites, and a foreign element of DNA is spliced into it.

What Are Plasmids?

Essentially, plasmids are small, circular molecules of DNA that are capable of replicating independently.

As such, they do not rely on chromosomal DNA of the organism for replication. Because they have this unique characteristic, they are often referred to as “extra-chromosomal DNA”.

While this type of molecule was initially discovered in one of the members of the Enterobacteriacae family, they also occur naturally in many different microorganisms throughout the world.

There have been many debates revolving around whether plasmids are truly microorganisms using the existing virus definition.

However, it’s important to note that the term “plasmid” is actually used primarily to refer to genetic elements that are capable of independently replicating.

These genetic elements also exist outside an organism’s chromosome.

Where Can They Be Found?

Plasmids can be found in a surprising number of organisms. These include:

  • Archaea (single-celled organisms similar to bacteria)
  • Bacteria (single-celled organisms)
  • Eukaryotes (including yeast and plants)

The Structure Of Plasmids

The structure of plasmids are a fascinating topic. They are made up of double circular chains of DNA.

This circular shape is only made possible because both ends of the double strands come together and are joined by covalent bonds.

These molecules are miniscule in size – this is especially the case if you were to compare their size to the DNA or organisms.

Plasmids can range in size and be anywhere from only a few kilobases to a few hundred kilobases.

While many plasmids have circular structures that are closed with covalent bonds, there are also plasmids that have linear shapes.

As such, these plasmids are not circular! When considering plasmids, there are three primary components which are included.

These components are as follows:

Replicon

Also known as the origin of replication (ori), this refers to the precise location in the strand where replication starts.

In plasmids, this location is primarily made up of A-T base pairs, which are easier to replicate during the replication process.

While other DNA have many origins of replication, when it comes to plasmids, they on;y have a select few. This is likely to be due to their tiny size.

These plasmids also have more regulatory elements, which contribute to the process of replication, such as replication proteins.

Polylinker

Also known as multiple cloning sites (mcs), the polylinker is actually considered to be one of the most crucial parts of the whole molecule. It’s incredibly important because it allows us to learn a lot about cloning.

In essence, a polylinker is a short sequence of DNA which consists of a select few sites where cleaving occurs due to restriction enzymes.

Because of this, the polylinker allows DNA to be inserted, and can restrict it when DNA is inserted.

Antibiotic Resistance Gene

This is one of the main parts of a plasmid. The antibiotic resistance gene plays a key role in drug resistance, as the name might suggest.

This ability makes the treatment of a number of diseases far more challenging, as a resistance is built up.

Plasmids are now known for their ability of conjugation. This refers to their ability to transfer themselves from one type of bacteria to another one.

During this process, they are able to confer antibiotic resistance properties to the bacteria that they transfer to, spreading immunity.

It’s also worth noting that, although plasmids have the added advantage of gaining resistance compared to bacteria, this also has an effect on bacteria cell division.

This is because of the additional burden they experience from replication. Because of this, the bacteria which have plasmids are often the minority compared to those that do not have plasmids.

This is due to the reduced cell division, which bacteria without plasmids would not have an issue with.

However, there are a further two components to include when thinking about plasmids. These are as follows:

  • Primer Binding Site – this is a short DNA sequence on a single strand. It is usually needed for things like DNA sequencing and PCR amplification.
  • Promoter Region – the promoter region is effectively in charge of recruiting transcriptional machinery.

Types Of Plasmids

While there are a number of plasmid shapes and characteristics, there are many plasmids in the world. These plasmids vary greatly, and all take on different features, characteristics, and shapes.

Let’s take a look at some different plasmids in the sections below.

Resistance Plasmids

Resistance plasmids, also called antimicrobial resistance plasmids, are a type of plasmids which play a key role in antibiotic resistance. They carry specific genes that allow them to have this ability.

These plasmids are also very involved in bacterial conjugation, as they produce conjugation pili. These pili are known to transfer the R plasmid onto one bacterium from another one.

There are two primary groups of resistance plasmids, which we will go through below:

  • Broad-Host-Range Group – These plasmids can easily be transferred from one species of bacteria to another. They can carry a wide range of antibiotic resistance genes. If plasmids with antibiotic resistance genes are transferred to drug-sensitive bacteria, the bacteria are likely to develop a resistance to the drug.
  • Narrow-Host-Range Group – These are typically replicated in one species.

Degradative Plasmids

Degradative plasmids have the ability to allow the host organism to break down to degrade xenobiotic compounds.

These compounds are also referred to as recalcitrant substances, and include a wide range of compounds that are released into the environment due to human actions.

Because of this, these compounds are not naturally occurring, and are not very common in nature.

You can find the hosts of degradative compounds in the groups IncP-1, IncO-7, and IncP-9. This includes many species, including Pseudomonas fluorescens, Rhizobium sp, Ochrobactrum anthropi, Escherichia coli, and Burkholderia hospita, among hundreds more.

Researchers have attempted to use these plasmids to degrade different contaminating substances within the environment, though this has not been successful. This process is known as Bioaugmentation.

Unfortunately, despite the ability for xenobiotic compounds to be hosted, these plasmids have been unsuccessful in cleaning up the environment.

However, research continues to be done, and work is still being done to discover ways to use indigenous bacteria to degrade these unnatural compounds.

A number of factors have an effect on the degradation of xenobiotic compounds – a behavior that could be useful. Factors such as stability and the capacity of replication are high on the list of important things to consider.

An example of this would be looking at the plasmids found in IncP-1 – a group that is known to have a high transfer frequency and also a broad host range.

The variations in behaviors of different degradative plasmids have then been linked to the different behaviors noted between their respective hosts and them.

Fertility Plasmids

Like a number of other plasmids, these ones, known as fertility plasmids (F plasmids), are also round. They have a circular structure, and typically measure around 100 kb.

There are four primary parts of a fertility plasmid, as follows:

  • Replication sites (RepFIA, RepFIB, and RepFIC)
  • Replication origin regions
  • Origin of conjugative transfer (oirT)
  • Transposable element (IS2, 1S3, and Tn1000)

These fertility plasmids play a key role in reproduction. This is because they contain genes that code for sex pilus and enzymes for conjugation in reproduction.

Not only that, but this plasmid also has genes that are needed in their own transfer in order to enhance their move from one cell to another.

The cells required to process fertility plasmids are called “donors”, while the cells without this ability are referred to as “recipients”.

Plasmids that have the ability to enhance the host cells’ ability to behave like a donor are called the “transfer factor”.

During the conjugation process, the donor cell, which is a bacterium, along with a sex pili (usually 1-3 pili), bind to a particular protein.

This binding happens on the recipient’s outer membrane, and initiates the whole mating process. Once the initial binding has taken place, the pili will retract and allow the two cells to bind together.

This is followed by the DNA transfer to the recipient from the donor, and ultimately the transfer of the fertility plasmid.

This results in the recipient acquiring the fertility plasmid, which allows them to create and produce sex pilus, which is involved in conjugation.

These tiny structures resemble rods, and allow the cells with F factor bacterial cells to attach themselves to the F-negative cells.

It’s important to note that, during conjugation, DNA is the only thing that is passed to the recipient from the donor. There is no other cell material such as cytoplasm that also gets transferred.

Col Plasmids

These plasmids are able to confer the ability to create toxic proteins (colicines) to bacteria. Bacteria including Salmonella, E. coli, and Shigella then use these toxins to kill the other bacteria, allowing them to thrive in their environments.

As there are a number of Col plasmids types, they are all able to produce different colicines or colicins.

Some examples of these include Col B, Col E2, and Col E3. The differences between the colicides are characterized by their various modes of action.

For example – while Col B is known to cause the cell membrane of other bacteria without plasmids to become damaged, Col E3 can induce degradation of a target cell’s nucleic acid.

Much like the fertility plasmids, a number of the Col plasmids have been known to carry various elements that enable them to enhance their transmission.

This is important when they move from one cell to another one, making them more successful.

As a result, Col plasmids are able to be transferred from a donor to a recipient through the conjugation process, typically with a cell with the fertility plasmids factor.

When conjugation occurs, the recipient is then able to produce the toxins required to inhibit or kill the bacteria they are targeting. However, this can only be successful if the targeted bacteria lack the plasmid.

Virulence Plasmids

Bacteria that are typically pathogenic in nature often carry genes for virulence factors. This virulence factor gives them the ability to invade and infect their hosts, which is not possible in harmless bacteria.

For a number of these bacteria, the organism’s genetic material results in the virulence factor. However, this is not always the case.

There can also be virulence factors because of extra-chromosomal DNA. There have also been other sources of elements like this, such as transposons, plasmids are among the most common mobile genetic elements.

When considering pathogenicity, virulence factors are very important. Their role is to aid the bacteria in effectively adapting to the environment, in order to thrive.

This type of plasmid has the ability to cause an organism to express various virulence-associated behaviors and functions.

When this happens, the organism gets more characteristics that are advantageous, which in turn allow them to do well in their new and challenging environment.

Virulence plasmids, like other types of plasmids, also have the ability to be transmitted to one bacterium from another.

Other than the virulence gene, plasmids are also known to carry important elements, which allow them to enhance transmission and improve maintenance.

Because of this, they are typically much larger size-wise, but there are fewer of them. This is thought to be the case so that they do not burden the organism when they go through cell division.

Both cell maintenance and well division typically require a lot of energy.

By the virulence plasmids being relatively low in numbers, they do not create a huge metabolic burden. If there were more of them, they would likely cause issues and not thrive.

Other Plasmids Include

There are five other plasmids that are worth mentioning before we continue, These plasmids are:

  • Cryptic Plasmids – Their functions are not yet known
  • Suicide Plasmids – They are not successful in replicating when they are transferred to one fell from another
  • Recombinant Plasmids – These plasmids have been altered within a laboratory and introduced to the bacteria for studies
  • Conjugative Plasmids – They promote the self-transfer of plasmids
  • Metabolic Plasmids – These plasmids enhance their host’s metabolism

Cloning

When it comes to cloning, there are two primary things you should know about. We will go through each of these below in detail to help you get a better understanding of them.

Plasmid Vector

This refers to any part or piece of a molecule which has genetic material that can be expressed once being replicated after transferal.

This definition makes it clear why the term “vector” and “plasmids” are frequently interchanged. However, it should be noted that not all plasmids are, in fact, vectors.

One major characteristic of a plasmid vector is their small size. They are very small, but apart from this tiny size, they also have an origin of replication.

On top of that, a vector will have a number of cloning sites and also a selective marker.

Ideally, a plasmid vector would have high copy numbers within the cell. This means that many targets will be cloned over time.

Additionally, this high copy number means that the gene of interest will be increased when genomes are divided.

In order to determine whether cloning was successful or not, the plasmid may also have a marker gene, which acts as a visual marker.

Since plasmids have a number of cloning sites, they are some of the best vectors when it comes to cloning.

Restriction enzymes have the ability to cleave different regions of the plasmid for cloning purposes, making them very useful.

As such, vectors have been used for a lot of research over the years. They are frequently used in studies looking at recombinant DNA being introduced into host cells.

This type of research has resulted in scientists being able to sequence the genomes of a number of various species.

Not only that, but also study the expression of different genes, and observe cellular mechanisms we would otherwise be unaware of.

Plasmid Isolation

Plasmid isolation has to be carried out before scientists can get purified plasmid DNA. This DNA would be used for processes such as cloning, transfection, and PCR.

The process of plasmid isolation involves the use of various techniques to obtain the DNA from the plasmid from host cells, and use it in molecular biology.

In order to carry out plasmid isolation, you will need to observe or carry out the following:

  1. Cell Growth – The bacteria containing the plasmid must grow in a shaken culture. Antibiotics might be used to prevent any kind of unwanted bacteria from growing, to keep the culture pure.
  2. Centrifugation – The bacterial growth gets followed by centrifugation. This is done to pellet cells. When the supernatant (clear liquid) has been removed, the process of isolating plasmids can start.

There are a number of methods that can be used to isolate plasmids. However, the most common and classical method that is used is known as alkaline analysis.

Alkaline analysis includes the following steps:

  1. Suspending The Pellet In An Isotonic Solution – The bacterial pellet that was obtained from centrifugation gets re-suspended in an ethylene diamine tetraacetate (isotonic) solution. This is done to prevent any activity within the nucleus.
  2. Alkaline Lysis Of The Cells – Cell lysis takes place using sodium dodecyl sulfate. This process disintegrates the cell membrane’s lipid structure.
  3. Precipitation Of The Dissolved Proteins – This is done using an acidic potassium acetate/
  4. Sedimentation – Centrifugation is then used for sedimentation
  5. Purification – A chloroform and phenol mixture is then used for the purification of the DNA from the plasmid. Here, the protein content is removed from the plasmid.
  6. Ethanol is added for precipitation for sedimentation through centrifugation
  7. The solution is then washed with 70% ethanol. This is done to remove the salt content of the mixture
  8. The DNA from the plasmid is then centrifuged again to sediment the mixture
  9. The sediment DNA from the plasmid is then dissolved in a Tris-EDTA or TW solution and stored for use.

Once plasmid isolation has taken place, and you have the pure plasmid DNA, it can be used for gene therapy, transfection, DNA sequencing, and, of course, cloning.

However, in order for these processes to work, the final product has to be a high quality, pure DNA from plasmids. If it is not, it will not be able to be used for such things, resulting in wasted time and money.

Final Thoughts

Plasmids are tiny molecules of DNA found in bacteria. While most of them are circular, there are linear types out there, too.

While plasmids don’t rely on chromosomal DNA of the living organism in order to replicate, they are essential in the cloning process, and have enabled scientists to explore a wide range of fields in molecular biology.

There is still much debate about whether plasmids should be considered as microorganisms.

However, the term “plasmid” is typically used to refer to the genetic elements that are capable of replicating and do not exist inside the DNA of an organism.

No matter what they end up being referred to, however, it is clear the plasmids are incredibly important in science, and are a fascinating subject.

The structure of plasmids can vary, as can their size and purpose. From Resistance plasmids to Fertility and Col plasmids, they have incredible importance.

This just shows that even the smallest things that are not visible to the human eye can have a huge impact on science and the world as we know it today.

Jennifer Dawkins

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