In biotechnology and molecular biology, the insertion of foreign DNA into bacterial cells is one of the most indispensable laboratory techniques. This technique, known as transformation, heavily relies on the preparation of competent cells. Competent cells are specially treated bacterial cells that readily absorb DNA, such as plasmid, from their surroundings, making them essential for molecular cloning, studies of gene expression and recombinant DNA technology.
What are competent cells?
Competent cells are bacteria (typically Escherichia coli or E.coli) that have been chemically induced to readily take up exogenous DNA from their environment. Under normal conditions, bacteria cannot easily assimilate DNA. But under a specific set of treatments the cell membranes of bacteria are made susceptible and to take up DNA from its environment. This is the process of competency induction, and is fundamental in the processes of genetic engineering.
Why are competent cells important?
Preparation of competent cells is a fundamental step in modern biotechnology. Without competent cells, most modern molecular biology research would not be possible. Competent cells facilitate:
* Gene cloning into plasmid vectors
* Recombinant protein expression
* Genetic transformation of organisms
* Functional studies of gene regulation and expression
* Development of therapeutics and vaccines
Competent cells simply act as a medium to transfer DNA molecules from the external environment to within a living organism.
Methods of Competent Cell Preparation
There are two principal methods by which competent cells are prepared:
1. Chemical method (Calcium Chloride method)
This is by far the most common and most efficient method for making competent cells, as it is the least expensive method.
Principle:
Cell membrane and DNA both bear negative charge, which causes repulsion between them. The presence of Calcium ions (Ca) effectively neutralize this charge and draws the DNA closer to the cell membrane, making it more susceptible.
Procedure:
1. Growing bacteria
Bacteria are grown in nutrient media (eg. LB media) to the middle log phase (OD of 0.4-0.6). At this phase, bacteria are most responsive and growing at their maximal rate.
2. Icing the culture
The culture is cooled down in the presence of ice and kept in the ice. This lowers the metabolic rate and stabilized the membranes of the cell.
3. Treatment with Calcium Chloride (CaCl)
Bacteria is mixed with the cold CaCl solution, causing the cell membranes to turn permeable to DNA.
4. Centrifuging and re-suspendation
The cells are centrifuged in the cold at a slow speed and suspended in another dose of fresh, cold CaCl solution.
5. Aliquoting and freezing
Bacteria cells are collected into small samples and snap frozen at -80 degrees C for further use.
6. Heat shock (Transformation procedure)
Upon transformation, the competent cells are mixed with the DNA and then placed on a heat block at 42 degrees C for 45-60 seconds. This thermal shock causes the entry of DNA into the cells.
2. Electroporation
In this procedure, DNA enters the cells when a strong electrical field is applied.
Principle:
A high-voltage pulse applied to the solution of DNA and cells creates pores in the cell membrane. The DNA can pass through these temporary pores into the cells.
Pros and Cons
* It gives very high transformation efficiency compared to the chemical method.
* Can be used to transform with larger DNA molecules.
However,
* It requires a more expensive piece of equipment to do so
Factors affecting competency
The competency efficiency of competent cells is affected by several factors, which can include:
* Cold temperatures are important when preparing the cells
* The exponential phase of cell growth should be maintained
* Pure plasmid DNA is used for transformation
* Accurate heat shock period
* Sterile environment
Small variations in any of these steps may lead to greatly reduced transformation rates.
Uses of competent cells
Competent cells are utilized in many areas within the biotechnology and molecular biology field:
1. Genetic Engineering
It is used for inserting desired gene fragments into plasmid vectors and then transforming a selected bacteria strain.
2. Recombinant protein production
Genes for desirable proteins such as insulin and growth hormones are cloned using competent cells in a specific bacteria strain.
3. Studying gene functions
They facilitate various genetic modification procedures to learn the function of a specific gene and its regulatory mechanisms.
4. Vaccine Development
Development of vaccines using a DNA molecule (DNA vaccine) may include Cloning a target DNA fragment using competent cells.
Advantages of using competent cells
* Very inexpensive to prepare, especially when using chemical method
* Easy and fast to prepare
* Can accommodate most cloning procedures and studies
* High transformation efficiency in suitable prepared cells
Drawbacks of competent cells
Competent cells do not pose an advantage as such; they simply enable the transformation process, and like any other procedure it does not come without challenges:
* Preparation efficiency is highly variable
* Certain bacterial strains do not respond to competent cell transformation procedures
* Over heating will kill the bacteria
* requires use of sterile tools
Practical notes on the preparation of competent cells from personal laboratory experience
On a practical note, incompetent cell preparation really allows one to discover the joy of patience and perfection in science. All aspects should be controlled very carefully. Maintaining the culture at cold conditions during preparations should be really consistent, otherwise even small amount of temperature fluctuation could be significant.
The reason that Calcium ions are used is that Calcium ions neutralize the negatively charged DNA and also negative charges on the cell membrane, making the DNA easy for entry. Heat shock will lead to a rapid change in temperature, the increase in cell membrane permeability is thought to cause DNA entry through transient pores generated in the membrane. The time of the heat shock should be carefully controlled; if it lasts too long then the cells could die and thus result in very poor transformation efficiency.
Conclusion
The preparation of competent cells is a very useful laboratory technique used by all the molecular biology and biotechnology researcher, scientist, doctor.. The use of these special bacteria cells allows scientists to insert specific foreign genes into cells and propagate them. Various different methods are available and used to prepare competent cells such as the chemical method using Calcium chloride, or the electroporation technique.
These methods can greatly aid scientific advancement within the fields of medicine, agriculture and industry. This technique has truly broadened the scope and range of what scientists are able to learn and discover, and makes many forms of experimentation readily available for researchers in the lab.
The field is continuing to evolve, so it is important to be knowledgeable on all of the useful techniques currently available for gene manipulation. Competent cells can be used to investigate gene function, create recombinant DNA molecules, or develop and test vaccines, the possibilities appear endless.
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