This protocol describes the preparation and transformation of competent E. To calculate the transformation efficiency, an indicator of how well the cells took up the extracellular DNA, the colonies obtained in the transformation need to be counted:. Table 1: Colony forming units cfu counted from transformation experiment.
Many parameters affect the transformation efficiency: plasmid size, cell genotype, growth stage during competence preparation, methods of transformation, etc. When calculating the TE is important to consider what dilution if any was performed before plating and incorporate it in the calculation of the total number of cfu.
The transformation efficiency TE is calculated with the following equation:. Then divide the result by the dilution factor. Bacteria are remarkably adaptable and one mechanism which facilitates this adaptation is their ability to take in external DNA molecules. One type of DNA that bacteria can uptake is called a plasmid, a circular piece of DNA that frequently contains useful information, such as antibiotic resistance genes.
The process of bacteria being modified by new genetic information incorporated from an external source is referred to as transformation.
Transformation can easily be performed in the laboratory using Escherichia coli, or E. In order to be transformed, E. The protocol for accomplishing this is surprisingly simple, a short incubation of the cells in a calcium chloride solution. This incubation causes the cells to become permeable to DNA molecules. After the cells are pelleted by centrifugation, the supernatant is removed. The plasmid DNA is now added to the competent cells. After incubating the cells with DNA, the mix is briefly heated to 42 degrees Celsius, followed by rapid cooling on ice.
This heat shock causes the DNA to be transferred across the cell's wall and membranes. The cells are then incubated in fresh media. Then, the bacteria are placed at 37 degrees to allow them to reseal their membranes and express resistant proteins. Those cells which have taken in the plasmids will faithfully copy the DNA and pass it to their progeny and express any proteins that might be encoded by it, including antibiotic resistance mediators. Those resistance genes can be used as selectable markers to identify bacteria which have been successfully transformed because cells that have not taken up the plasmid will not express the resistance gene product.
This means that when the cells are plated on a solid medium which contains the appropriate antibiotic, only cells that have taken up the plasmid will grow. Transformation of the cells in a growing colony can be further confirmed by culturing those cells in liquid media overnight to increase the yield before extracting the DNA from the sample.
Once the DNA is isolated, a diagnostic restriction enzyme digest can be carried out. Because restriction enzymes cut DNA in predictable locations, running these digests on a gel should show a predictable pattern if the desired plasmid was successfully transformed. For example, if pUC19 is prepared and cut with the restriction enzyme HindIII, a single band of nucleotides should be seen on the gel. In this lab, you will transform E. Before starting the procedure, put on the appropriate personal protective equipment, including a lab coat and gloves.
Now, prepare chemically competent cells by depositing a loopfull of bacteria onto a sterile LB agar plate and streaking the bacteria with a new loop.
Then, incubate the plate at 37 degrees Celsius overnight. Inoculate a single, well-isolated colony into 3 milliliters of LB broth in a tube with a sterile loop. Then, grow the culture at 37 degrees Celsius overnight, with shaking at RPM. The next day, measure the optical density of the overnight culture with a spectrophotometer. Then, add milliliters of LB broth to a one-liter flask, and inoculate it with the overnight culture at an optical density of 0. Now, incubate the culture at 37 degrees Celsius with shaking, and check the OD every 15 to 20 minutes until the culture reaches mid-exponential growth phase.
After approximately three hours, transfer 50 milliliters of the culture to two ice-cold polypropylene bottles. Then, place the bottles back on ice for 20 minutes to cool. Next, recover the cells via centrifugation. Discard the supernatants and place the bottles upside down on a paper towel. Next, resuspend the bacterial pellet in five milliliters of ice-cold calcium chloride magnesium chloride solution and swirl carefully until the pellet has dissolved completely.
Then, add another 25 milliliters of the solution to the dissolved bacterial pellet. Resuspend the other bacterial pellet as previously demonstrated. After this, repeat the centrifugation, and remove the supernatants. If the competent cells are going to be directly transformed, resuspend each bacterial pellet in two milliliters of an ice-cold 0.
To begin the transformation procedure, transfer 50 microliters of competent cells to two labeled 1. Mix gently, avoiding bubble formation, and incubate both tubes for 30 minutes on ice.
After incubation, transfer the tubes to a heat block and incubate at 42 degrees Celsius for 45 seconds. Biophysical journal , 93 6 , Panja, S. How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli?. Molecular membrane biology , 25 5 , Qiu, H. Adaptation through horizontal gene transfer in the cryptoendolithic red alga Galdieria phlegrea. Current Biology , 23 19 , RR Rahimzadeh, M. Impact of heat shock step on bacterial transformation efficiency.
Molecular biology research communications , 5 4 , Steinmoen, H. Induction of natural competence in Streptococcus pneumoniae triggers lysis and DNA release from a subfraction of the cell population. Proceedings of the National Academy of Sciences , 99 11 , The Gel Phase. Transformation, B. The Heat Shock Method.
Basic Methods in Cellular and Molecular Biology. Tsong, T. Electroporation of Cell Membranes. Electroporation and Electrofusion in Cell Biology , 60 , — Introduction to Competent Cells by Katharine Martin. What are competent cells? Competence vs. Transformation — The action of a competent cell taking up genetic material. Article Contents: What are 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 What is the difference between natural and artificial competent cells? Competent cells can either occur naturally or cells can artificially be made competent.
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.
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.
Therefore, in order to use these cells for cloning, researchers must induce competence. What exactly makes a cell competent? 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.
List of salts and chemicals used to make chemically competent cells CaCl 2 : Neutralizes the negative charges of the phospholipid bilayer and DNA. A look at transformation efficiencies in E.
Google Scholar. Curtiss, R. III, Inoue, M. Scott and R. Dagert, M. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6, 23— Day, M. Miller and M. Dower, W. High efficiency transformation of E. Nucleic Acids Res. Dreiseikelmann, B.
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Gene 25, — Kushner, S. Amsterdam: Elsevier. Lacks, S. London: Chapman and Hall. Li, W. Exploring the mechanism of competence development in Escherichia coli using quantum dots as fluorescent probes. Methods 58, 59— Liu, C. Conducting nanosponge electroporation for affordable and high-efficiency disinfection of bacteria and viruses in water. Nano Lett. Mandel, M. Calcium-dependent bacteriophage DNA infection. AA and HM drafted the manuscript.
YR put forward the idea of the manuscript and edited the manuscript to the final form. RT helped in the write up of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
National Center for Biotechnology Information , U. Journal List Front Microbiol v. Front Microbiol. Published online Nov 7. Author information Article notes Copyright and License information Disclaimer. This article was submitted to Microbial Physiology and Metabolism, a section of the journal Frontiers in Microbiology. Received Jul 18; Accepted Oct Keywords: calcium chloride, transformation, Escherichia coli , DNA, competent cells.
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This article has been cited by other articles in PMC. Open in a separate window. Figure 1. Author contributions AA and HM drafted the manuscript. Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Blattner F. Charon phages: safer derivatives of bacteriophage lambda for DNA cloning. Science , — Construction and characterization of new cloning vehicle.
A multipurpose cloning system. Gene 2 , 95— A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. A look at transformation efficiencies in E. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 6 , 23— Transformation , in Microbial Evolution , eds Miller R. High efficiency transformation of E. Nucleic Acids Res.
Translocation of DNA across bacterial membranes. Genetic studies with heteroduplex DNA of bacteriophage f1.
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