1: Bacterial Transformation
Bacterial transformation is the process by which bacteria take up free DNA from their surroundings. This process is commonly used to make bacteria take up and replicate recombinant DNA during molecular cloning.
Also known as
Transformation
Samples needed
Sample of DNA (typically recombinant DNA) and live bacterial cells
Method
Bacterial transformation is a molecular biology technique that utilizes the replication system of bacteria to replicate recombinant DNA. Bacterial transformation involves a 4 step process:
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Generation of competent cells
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Introduction and transformation of the DNA into the bacteria
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Recovery
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Plating
In step 1, competent cells are bacterial cells that are able to efficiently take up free DNA from their environment. Bacteria can be made competent through a variety of techniques such as incubation of the bacteria in a solution containing ions (such as calcium) that are thought to play a role in DNA adhering to the cell membrane and making it easier for DNA to enter during step 2.
In step 2, the DNA sample is added to the competent bacteria. The bacteria + DNA are then briefly exposed to a high temperature, a process known as heat shock. This allows for uptake of the DNA by the competent bacteria. Alternatively, a process known as electroporation may be used, where the bacteria are briefly subjected to a high-voltage electric field that creates pores in the cell membrane that allow for entry of the DNA.
In step 3, the bacteria are placed in liquid growth media and given time to recover from any cell membrane disruption that occurred during the heat shock or electroporation process. This also provides time for the bacteria to synthesize any enzymes encoded by the DNA, such as enzymes conferring antibiotic resistance.
In step 4, the bacteria are spread on plates of solid growth media and allowed to form colonies. Typically this growth media will contain antibiotics to help determine which bacteria have taken up the DNA. This antibiotic selection works by having an enzyme conferring antibiotic resistance encoded by the DNA sample so that only bacteria that took up the DNA will be able to survive and form colonies on the media containing antibiotics.
Controls
A no DNA control (water instead of DNA) is typically included as a negative control to ensure that no reagents are contaminated. Known plasmid DNA with the desired antibiotic resistance can serve as a positive control. Researchers will also sometimes include a comparison of colony growth between antibiotic and non-antibiotic containing media to verify that antibiotic selection occurred.
Interpretation
The authors sought to optimize bacterial transformation of Clostridium perfringens , a bacteria known to cause foodborne illness. In this experiment, the researchers first measured the ability to transform plasmid DNA into strains of C. perfringens known to cause foodborne illness (NCTC8239, W4232, W5837, and W09-505) compared to the better characterized SM101 strain. The researchers mixed plasmid DNA with competent C. perfringens cells, utilized the electroporation technique to transform plasmid DNA into cells, and determined the number of colonies on media containing the antibiotic erythromycin. The efficiency of the transformation was then calculated by dividing the number of colonies by the amount of DNA (in micrograms) and the amount of viable bacteria that were plated. The W4232 strain showed a similar transformation efficiency to the SM101 strain, while the NCTC8239 and W5837 strains showed significantly lower numbers of transformed bacteria compared to SM101. Notably, no transformed bacteria were observed for W09-505. Therefore, three out of the four strains tested showed decreased ability to uptake DNA compared to the SM101 strain.
Image Descriptions
Figure 1 image description:
A bar graph. The bar graph shows the number of transformant bacteria divided by micrograms of DNA divided by viable cells. SM101 and W4232 are both at 10 -4 , while NCTC8239 is at 10 -7 and W5837 is at 10 -5 . W09-505 has no bar on the graph and instead says ND. ↵
Thumbnail
"Vibrio cholerae on TCBS agar.jpg" ↗ by Microrao is in the Public Domain ↗.
Image description: Vibrio cholerae on TCBS agar
Author
Rachel Ende, Tufts University
1. Yasugi, M, D. Motooka, S. Nakamura, and M. Miyake. 2020. Phosphorothioation of foreign DNA influences the transformation efficiency in Clostridium perfringens NCTC8239. Anaerobe 61:e102085. ↵