Informational

TD-I Revision 1.0 Creation Date: 6/15/2020

Revision Date: 7/10/2020

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.

Article Contents:
What are competent cells?

What is the difference between natural and artificial competent cells?

Natural cell competence:

Artificial (induced) cell competence

What exactly makes a cell competent?

Types of artificially competent cells

Chemically competent cells

Electrocompetent cells

References

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What are competent cells?

Cell competence refers to a cell’s ability to take up foreign (extracellular) DNA from its
surrounding environment. The process of genetic uptake is referred to as
transformation. In some cases, the genetic material taken in by a cell can become
incorporated, or recombined, into its own genome. This incorporation of genetic material
into the host genome is called horizontal gene transfer (HGT) or lateral gene transfer.

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.

What is the difference between natural and

artificial competent cells?
Competent cells can either occur naturally or cells can artificially be made competent.

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Natural cell competence:

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.

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Artificial (induced) cell competence

Artificial or induced competent cells are cells researchers have made competent
through electrical (electroporation) or chemical manipulation. Cell competence has
become an essential research tool for cloning because it provides scientist a
mechanism to introduce new genetic material into a cell. When we think of competent
cells in a research setting, this is often the type of cell competence being referred to.
While there are many naturally occurring competent cells, the model organism E. coli is
not naturally competent. Therefore, in order to use these cells for cloning, researchers
must induce competence.

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.

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What exactly makes a cell competent?

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.

Types of artificially competent cells

There are two types of artificially competent cells: chemically competent and
electrocompetent.

Chemically competent cells

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.

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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.

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Electrocompetent cells

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.

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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

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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

References
Blokesch, M. (2016). Natural competence for transformation. Current Biology, 26(21),
R1126-R1130.

Chan, W. T., Verma, C. S., Lane, D. P., & Gan, S. K. E. (2013). A comparison and
optimization of methods and factors affecting the transformation of Escherichia
coli. Bioscience reports, 33(6), e00086.

Chen, I., & Gotschlich, E. C. (2001). ComE, a competence protein from Neisseria
gonorrhoeae with DNA-binding activity. Journal of bacteriology, 183(10), 3160-3168.

Cheng, C. Y., Song, J., Pas, J., Meijer, L. H., & Han, S. (2015). DMSO induces
dehydration near lipid membrane surfaces. Biophysical journal, 109(2), 330-339.

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Deipolyi, A. R., Golberg, A., Yarmush, M. L., Arellano, R. S., & Oklu, R. (2014).
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oncology. Diagnostic and Interventional Radiology, 20(2), 147.

Dybvig, K., Gasparich, G. E., & King, K. W. (1995). Artificial transformation of mollicutes
via polyethylene glycol-and electroporation-mediated methods. Molecular and
diagnostic procedures in mycoplasmology, 1, 179-184.

Finkel, S. E., & Kolter, R. (2001). DNA as a nutrient: novel role for bacterial competence
gene homologs. Journal of Bacteriology, 183(21), 6288-6293.

Hanahan, D., Jessee, J., & Bloom, F. R. (1991). [4] Plasmid transformation of
Escherichia coli and other bacteria. In Methods in enzymology (Vol. 204, pp. 63-113).
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Jain, R., Rivera, M. C., & Lake, J. A. (1999). Horizontal gene transfer among genomes:
the complexity hypothesis. Proceedings of the National Academy of Sciences, 96(7),
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LabBench Activity: Competent Cells. (n.d.). Retrieved December 11, 2019, from
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Mell, J. C., & Redfield, R. J. (2014). Natural competence and the evolution of DNA
uptake specificity. Journal of bacteriology, 196(8), 1471-1483.

Napotnik, T. B., & Miklavčič, D. (2018). In vitro electroporation detection methods–An

overview. Bioelectrochemistry, 120, 166-182.

Narayanan, G. (2015, December). Irreversible electroporation. In Seminars in

interventional radiology (Vol. 32, No. 04, pp. 349-355). Thieme Medical Publishers.

Notman, R., den Otter, W. K., Noro, M. G., Briels, W. J., & Anwar, J. (2007). The
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molecular dynamics. Biophysical journal, 93(6), 2056-2068.

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Web: www.goldbio.com 10
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Panja, S., Aich, P., Jana, B., & Basu, T. (2008). How does plasmid DNA penetrate cell
membranes in artificial transformation process of Escherichia coli?. Molecular
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Qiu, H., Price, D. C., Weber, A. P., Reeb, V., Yang, E. C., Lee, J. M., ... & Bhattacharya,
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Rahimzadeh, M., Sadeghizadeh, M., Najafi, F., Arab, S., & Mobasheri, H. (2016).
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Steinmoen, H., Knutsen, E., & Håvarstein, L. S. (2002). Induction of natural competence
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cell population. Proceedings of the National Academy of Sciences, 99(11), 7681-7686.

The Gel Phase. (2015, April 9). Retrieved December 11, 2019, from Physics LibreTexts
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_Membrane_Biology/3%3A_Membrane_Phases_and_Morphologies/3.4%3A_The_Gel_Ph
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