If the problem continues, please let us know and we'll try to help. An unexpected error occurred. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the creation of pores in bacterial cell membranes. Bacterial cells that are able to take up DNA from the environment are called competent cells. In the laboratory, bacterial cells can be made competent and DNA subsequently introduced by a procedure called the heat shock method.
Heat shock transformation uses a calcium rich environment provided by calcium chloride to counteract the electrostatic repulsion between the plasmid DNA and bacterial cellular membrane. A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell.
This video goes through a step-by-step procedure on how to create chemically competent bacteria, perform heat shock transformation, plate the transformed bacteria, and calculate transformation efficiency. Bacterial transformation is a widely used method where foreign DNA is introduced into a bacterium, which can then amplify, or clone the DNA.
Cells that have the ability to readily take up this DNA are called competent cells. In this video we will talk about one of these ways, heat shock transformation. A plasmid is a small, circular, double-stranded DNA that can reduce its size by supercoiling, so that it can easily pass through pores in a cell membrane. A plasmid contains a few important regions worth mentioning. Commercially available plasmids contain a multiple cloning site or MCS.
This region contains specific sequences recognized by restriction endonucleases or restriction enzymes, which cleave DNA. DNA fragments of interest to the researcher can be inserted into the multiple cloning site when the plasmid and DNA fragment are cut with the same endonucleases. Plasmids also contain an Origin of Replication, or ORI, that provides information to the cell as to where replication of the plasmid should begin.
In addition to the origin of replication and the multiple cloning site, most plasmids will include an antibiotic resistance gene.
This gene confers antibiotic resistance to all cells that contain the plasmid, allowing those cells to survive in antibiotic-containing media. The most commonly used type of bacteria in molecular biology research, and transformation is E. Cells are typically made competent via exposure to a calcium rich environment. The positive charges of the calcium ions neutralize the negative charges of both the plasmid and the bacterial cell wall dissipating electrostatic repulsion and weakening the cell wall.
By exposing cells to a sudden increase in temperature, or heat shock, a pressure difference between the outside and the inside of the cell is created, that induces the formation of pores, through which supercoiled plasmid DNA can enter.
After returning the cells to a more normal temperature, the cell wall will self-heal. Once cells have taken up the plasmid, they will be able to grow on agar plates laced with antibiotic.
Before starting heat shock transformation, clean the work area and make sure all equipment is sterilized. Ensure that you have enough media and agar prepared, which provide the nutrition to the bacteria you will make competent. Also be sure to sterilize all solutions via autoclaving. Allow plates to cool to room temperature to solidify. When working with bacteria, one should always use aseptic technique to maintain sterility.
Aseptic technique typically involves the use of a Bunsen burner to sterilize instruments and reagents and create a convection current — which keeps airborne contaminants out of the workspace.
Using proper aseptic technique, add uL bacteria to an LB agar plate and spread the medium around with a bacterial spreader. Next, count the colonies to calculate the transformation efficiency, which is the number of successful transformants divided by the total amount of DNA plated. This article provides information about how to use competent cells, types of competent cells, the common steps to make competent cells, and competent cell storage.
The preparation step : the bacterial cells are made competent to uptake foreign DNA by modifying the permeability of the cell membrane and the cell wall. The most common transformation methods are electroporation or heat shock transformation.
The recovery step : the cells are incubated in a recovery medium to restore the cell membrane and the cell wall. The advantages of using electroporation are the higher efficiency, more colonies, and much faster transformations compared to heat shock method.
Transformation efficiencies for electroporation are 5. Therefore, electroporation is helpful when you have to construct DNA libraries. The drawback of this method is that you must have an electroporator, which is a special piece of equipment. In addition, the common problem during electroporation is the presence of salts or air bubbles in your DNA, and in the cuvette, can cause an arcing.
Unfortunately, this will make you lose your sample and require you to redo your ligation reaction. Heat shock transformation is relatively easy compared to electroporation. It is also simple, only requiring a water bath.
You can use this method, when you only need to get a few positive clones. To perform transformation, you must have competent cells. There are two types of artificially competent cells available: electrocompetent and chemically competent.
What you use for electroporation is electrocompetent cells, whereas chemically competent cells are used for the heat-shock transformation method. Competent cells are bacterial cells commonly used for transformation. Transformation of bacteria involves the binding of foreign DNA to the cell membrane, and the movement of DNA across the membrane into the cytoplasm.
In electroporation, an electric pulse creates pores and a temporary electric field. The electric field pulls the DNA to the more positively charged end or into the cell. Preparing electrocompetent cells are relatively easier than making chemically competent cells. Glycerol, used for extensive washing, removes remaining salts from the pellet suspension. During the heat shock transformation, the heat pulse decreases the membrane potential of the competent cells, therefore lowering the potential barrier for the movement of negatively charged DNA into the cytoplasm Panja et al.
To make chemically competent cells, pellets are usually treated with salts, for example by using CaCl 2 or MgCl 2. This salt treatment neutralizes the negative charges of the phospholipid bilayer and DNA, allowing DNA to move closer to the cell. Introduction to Competent Cells. GoldBio competent cells are shipped on dry ice. Before use, thaw and keep competent cells on ice. Incubate the thawed cells with a plasmid DNA on ice for 30 minutes prior to transformation or a particular time suggested by your protocol to achieve optimal transformation efficiency Liu et al.
The competent cell preparation ahead of transformation must be kept at low temperature. It was suggested that heat shock step could facilitate DNA entry but still there is not enough clues. Panja et al reported that heat-pulse step cause reduction in membrane potential [ 2 ].
The cellular inside potential is became less negative as a result of membrane potential decrease therefore, the negative DNA could enter the cytosol easier [ 2 ]. The authors declare that there is no conflict of interests. National Center for Biotechnology Information , U. Mol Biol Res Commun. Author information Copyright and License information Disclaimer. Copyright notice. This article has been cited by other articles in PMC.
Abstract CaCl 2 treatment followed by heat shock is the most common method for artificial transformation. Key Words: E. Open in a separate window. Figure 1. Figure 2. Conflict of Interest: The authors declare no conflict of interest. References 1. Chen I, Dubnau D. DNA uptake during bacterial transformation. Nat Rev Microbiol. Role of membrane potential on artificial transformation of E coli with plasmid DNA. J Biotechnol. Bacterial transformation using micro-shock waves.
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