This article offers an in-depth introduction to competent cells along with providing helpful illustrations. Learn about what competent cells are, what the difference is between natural and artificial competence, how cells are made competent, how electroporation and calcium chloride treatment work, and so much more.
Competence vs. Transformation Competence – A cell’s ability to take up extracellular DNA from its environment. Transformation – The action of a competent cell taking up genetic material.
Horizontal gene transfer (HGT) is more difficult in eukaryotic cells than prokaryotic cells because genetic material must get through both the cell membrane and the nuclear membrane. Because prokaryotic cells do not have membrane bound nuclei, DNA can integrate within a bacterial genome much easier.
Natural competence and gene transfer have facilitated many adaptations in prokaryotic and eukaryotic cells. One example of such adaptation is the eukaryotic red algae Galdieria sulphuraria. Gene transfer to this organism living in extreme environments came from prokaryotes via HGT. The transferred genes led to a more versatile metabolism and the ability to detoxify mercury and arsenic.
Competent cells can either occur naturally or cells can artificially be made competent.
Natural cell competence is genetically determined, that is to say, a bacterium is genetically predisposed to take up free genetic material (genetically competent) that exists within their environment. Research has shown that competent bacterial cells can take in DNA from a variety of sources; however, some bacteria show specificity or a preference for DNA fragments that are overrepresented within their genome.
DNA taken up by a naturally competent cell does not always become incorporated into the cell’s genome. Often, a fragment of DNA will be used for nutritional purposes. For example, DNA provides a cell a much-needed source of deoxyribonucleotides for replication. The determination of how DNA is used within a cell depends usually on the needs of the cell. Other factors include existing DNA damage within the cell and recombination ability of incoming DNA.
While a cell might be considered naturally competent, the state of competence is not necessarily a constant state. In fact, natural regulation of competence and transformation is important for protecting a cell. Regulation often occurs through environmental and biochemical signals.
For example, Streptococcus pneumoniae is a naturally competent bacterium; however, its competence is prompted by quorum sensing, or detecting and responding to cell population density through gene expression. For natural transformation to occur, there must be donor DNA present in the environment. One study determined that when conditions were met, donor DNA came from a “sub-fraction” of the S. pneumonia population, while the other portion was competent, taking up the DNA.
Whether through electroporation or chemical methods such as calcium chloride, the process of making competent cells creates temporary pores in a cell’s membrane in order for DNA to pass through. Recall, competence is a cell’s ability to take up foreign DNA from its environment, while transformation is the actual process of DNA uptake. Therefore, in order for a scientist to transform a cell, she must first make that cell competent. This is done by changing the cell in such a way that enables DNA to easily travel through the cell membrane.
Since natural competence is genetically determined, these cells will be equipped with special machinery to facilitate natural transformation, and often will have biochemical and environmental signals to regulate competency.
In a lab setting, usually with E. coli, artificial cell competence is made possible through a chemical process or through electroporation. Both of these methods alter the cell membrane, creating temporary pores that allow DNA to enter the cell.
There are two types of artificially competent cells: chemically competent and electrocompetent.
Chemically competent cells are cells that were made competent with a salt treatment followed by a heat-shock step. This process permeabilizes the cell membrane, allowing plasmid entry. Protocols using CaCl2 or MgCl2 are the most common method for making chemically competent cells, but some protocols involve other salts or combinations of various salts and chemicals.
List of salts and chemicals used to make chemically competent cells
- CaCl2: Neutralizes the negative charges of the phospholipid bilayer and DNA.
- DMSO: DMSO gathers at the hydrophilic heads of the lipid bilayer, weakening the forces. As DMSO concentration increases, the thickness of the bilayer decreases, increasing membrane permeability.
- MgCl2: Works the same way as CaCl2, but allows better DNA binding to the cell.
- PEG: Shields the negative charges of the phospholipid bilayer and DNA.
- RbCl: Works the same way as CaCl2 and MgCl2. Some researchers prefer RbCl when higher competency is required.
What role does CaCl2 and heat-shock treatment play in making competent cells?
When making chemically competent cells, the first step involves using a salt, typically CaCl2 or MgCl2. The salt (chemical) treatment neutralizes the negative charges of the phosphate heads and the negatively charged DNA. Neutralizing these charges eliminates the natural repulsion, allowing DNA to move closer to the cell.
The heat-shock step involves quickly cooling and heating cells which leads to temporary pores that allow DNA to enter the cell. The mechanism behind how this truly works is not well understood. One study suggests the heat-pulse step causes E. coli to release lipids from its surface, causing temporary pores that allow DNA entry into the cell.
Electrocompetent cells are made competent using an electrical pulse from an electroporator to create temporary pores (poration) in the cell membrane of either prokaryotic or eukaryotic cells. The electric pulse disrupts the cell membrane, causing slight realignment of the lipid bilayer, which allows exogenous material entrance into the cell. While exogenous material can enter the cell due to increased permeability, cellular material can also be lost during the process.
Researchers choose to use electroporation for many different reasons. Higher transformation efficiency from this method compared to other methods is one reason for the choice. Another reason researchers might use electroporation is to introduce other material, such as new drugs or molecular probes, into the cell.
How does electroporation work?
The phospholipid bilayer are dual chains of phospholipids (phospholipids are composed of a hydrophilic head and a hydrophobic tail). The hydrophilic heads of the bilayer face outward: one row faces the extracellular space, while the other row faces the intracellular fluid. The hydrophobic tails face each other inward, that is to say, the internal portion of the bilayer. When voltage from electroporation is applied, the electrical pulse rearranges the orientation of the bilayer in a way that forms a gap. Refer to the illustration below.
The difference between reversible and irreversible electroporation
Reversible electroporation (RE) involves a voltage of up to 1 kV. Cells that undergo reversible electroporation can survive the effects of membrane permeation and recover (the cell membrane can reseal afterward). Molecular biologists use this technique to temporarily permeate the cell membrane in order to introduce foreign molecular material, such as DNA, into a cell.
Irreversible electroporation (IRE) involves up to 3 kV of DC current. Unlike reversible electroporation where membrane nanopores are temporary, irreversible electroporation creates permanent pores that ultimately leads to apoptosis (programmed cell death). Researchers have been using IRE for tumor treatment. Therefore, IRE is not going to be a method used for molecular transformation and transfection.
For more information about competent cells and transformation, browse resources found on GoldBio’s competent cell page found here: https://www.goldbio.com/collection/competent-cells
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