Bacterial Transformation
Background
In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccin…
Back in the early 20th century, a British bacteriologist named Frederick Griffith was working with the pneumonia-causing bacteria Streptococcus pneumoniae. He performed a simple experiment using two different strains. One strain is known as the S strain because of a protective capsule that makes the colonies or clumps it forms appear smooth, and also makes it virulent or harmful. The second was the R strain, a version of the bacteria lacking the protective capsule, giving the colonies a rough appearance and rendering it non-virulent.
First, Griffith took some of the S strain bacteria and heated it, producing a heat-killed version of the S strain. Then, he gathered some mice and divided them into four groups. He injected the first group with the virulent S strain, and the second with the non-virulent R strain. He dosed the third group with the heat-killed S strain, and finally, be combined the heat-killed S strain and the R strain together, and injected this mix into the fourth group. As expected, the mice in the first group died, and the ones in the second and third group lived. But to Griffith's surprise, the mice in the last group also died.
When he published his study in 1928, he called this mysterious process transformation, as he speculated the presence of an underlying transforming principle, which allowed the previously non-virulent strain to become deadly. Later, in 1943, Avert, MacLeod, and McCarty reported that this transforming principle was likely desoxyribonucleic acid, or DNA, which we now know is the heredity material. Essentially what happened in Griffith's experiment was that when the bacteria were combined, some DNA leaked from the heat-killed S strain and into the R strain cells, transforming these non-virulent bacteria and passing on the information to make the protective capsule, turning the R strain into a virulent one, now able to kill the animals in the fourth group.
Fast forward to today, and scientists have developed a much simpler way to study bacterial transformation, using the bacteria E. coli and small, circular loops of DNA called plasmids. Usually, the plasmid used in transformation experiments includes a gene for a special function, like antibiotic resistance. E. coli are an excellent subject for transformation because they can display a property called competence, the ability to take up DNA from the environment. Essentially, this means that under certain environmental conditions, like a chemical or an electrical or heat shock, the cell wall of the E. coli can become temporarily permeable and allow the uptake of DNA from the environment. Once the plasmid is inside the E. coli, it can either hang out in the cytoplasm of its new host cell and be replicated and expressed over generations alongside the genome, or it may fully incorporate itself into the genome of the host. If the bacteria lose the plasmid at any point, the cell will also lose its antibiotic resistance, and so scientists grow the bacteria in a media containing the antibiotic to ensure that the only survivors are those containing the plasmid of interest.
In this lab, you will learn how to transform E. coli cells with a plasmid containing an antibiotic resistance gene, while practicing sterile microbiological techniques.
Back in the early 20th century, a British bacteriologist named Frederick Griffith was working with the pneumonia-causing bacteria Streptococcus pneumoniae. He performed a simple experiment using two different strains. One strain is known as the S strain because of a protective capsule that makes the colonies or clumps it forms appear smooth, and also makes it virulent or harmful. The second was the R strain, a version of the bacteria lacking the protective capsule, giving the colonies a rough appearance and rendering it non-virulent.
First, Griffith took some of the S strain bacteria and heated it, producing a heat-killed version of the S strain. Then, he gathered some mice and divided them into four groups. He injected the first group with the virulent S strain, and the second with the non-virulent R strain. He dosed the third group with the heat-killed S strain, and finally, be combined the heat-killed S strain and the R strain together, and injected this mix into the fourth group. As expected, the mice in the first group died, and the ones in the second and third group lived. But to Griffith's surprise, the mice in the last group also died.
When he published his study in 1928, he called this mysterious process transformation, as he speculated the presence of an underlying transforming principle, which allowed the previously non-virulent strain to become deadly. Later, in 1943, Avert, MacLeod, and McCarty reported that this transforming principle was likely desoxyribonucleic acid, or DNA, which we now know is the heredity material. Essentially what happened in Griffith's experiment was that when the bacteria were combined, some DNA leaked from the heat-killed S strain and into the R strain cells, transforming these non-virulent bacteria and passing on the information to make the protective capsule, turning the R strain into a virulent one, now able to kill the animals in the fourth group.
Fast forward to today, and scientists have developed a much simpler way to study bacterial transformation, using the bacteria E. coli and small, circular loops of DNA called plasmids. Usually, the plasmid used in transformation experiments includes a gene for a special function, like antibiotic resistance. E. coli are an excellent subject for transformation because they can display a property called competence, the ability to take up DNA from the environment. Essentially, this means that under certain environmental conditions, like a chemical or an electrical or heat shock, the cell wall of the E. coli can become temporarily permeable and allow the uptake of DNA from the environment. Once the plasmid is inside the E. coli, it can either hang out in the cytoplasm of its new host cell and be replicated and expressed over generations alongside the genome, or it may fully incorporate itself into the genome of the host. If the bacteria lose the plasmid at any point, the cell will also lose its antibiotic resistance, and so scientists grow the bacteria in a media containing the antibiotic to ensure that the only survivors are those containing the plasmid of interest.
In this lab, you will learn how to transform E. coli cells with a plasmid containing an antibiotic resistance gene, while practicing sterile microbiological techniques.
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Q1: What was Frederick Griffith's transformation experiment and why was it significant?
Griffith studied two Streptococcus pneumoniae strains: the virulent S strain with a protective capsule and the non-virulent R strain without it. When he combined heat-killed S strain with live R strain and injected mice, they died unexpectedly. This discovery of bacterial transformation in 1928 revealed that genetic material could transfer between bacteria, fundamentally changing our understanding of heredity and laying the foundation for modern genetic engineering.
Q2: How did scientists identify DNA as the transforming principle in Griffith's experiment?
In 1943, Avery, MacLeod, and McCarty demonstrated that the transforming principle was desoxyribonucleic acid, or DNA, the heredity material. They showed that DNA leaked from heat-killed S strain bacteria into R strain cells, carrying genetic information that encoded the protective capsule. This discovery proved DNA was responsible for passing hereditary traits between bacterial cells.
Q3: What are plasmids and why are they useful for bacterial transformation in the laboratory?
Plasmids are small, circular loops of double-stranded DNA found in bacterial cytoplasm that replicate independently from chromosomal DNA. They often carry genes for beneficial traits like antibiotic resistance. Scientists use plasmids as vectors to introduce foreign DNA into bacteria like E. coli because bacteria can easily take in exogenous genetic material and rapidly amplify it, making plasmids ideal tools for genetic engineering research.
Q4: What is bacterial competence and how does it enable DNA uptake?
Competence is the ability of bacteria like E. coli to take up DNA from their environment. Under specific conditions—such as chemical treatment with calcium chloride, electrical shock, or heat shock—the bacterial cell wall becomes temporarily permeable. This allows plasmids and other DNA molecules to cross the membrane and enter the cell, where they can be replicated and expressed alongside the bacterial genome.
Q5: Why do scientists grow transformed bacteria on antibiotic-containing media?
Scientists use antibiotic-containing media as a selection method to identify successfully transformed cells. Plasmids typically carry antibiotic resistance genes, so only bacteria that have taken up the plasmid survive on these plates. Non-transformed bacteria lack the resistance gene and die, ensuring that visible colonies contain the plasmid of interest and have been successfully transformed.
Q6: What happens to a transformed bacterium if it loses its plasmid?
If a transformed bacterium loses its plasmid, it also loses the genes carried on that plasmid, including antibiotic resistance. The cell reverts to its original phenotype and can no longer survive on antibiotic-containing media. This is why continuous selection on antibiotic plates is necessary to maintain populations of successfully transformed bacteria carrying the plasmid of interest.
Q7: How does bacterial transformation using plasmids differ from Griffith's original experiment?
Griffith's experiment involved natural DNA transfer between bacterial strains, while modern bacterial transformation using plasmids is a controlled laboratory process. Scientists artificially introduce recombinant plasmids containing specific genes into competent E. coli cells using heat shock or chemical treatment. This allows precise genetic manipulation and is the foundation for bacterial transformation using plasmids procedure in contemporary genetic engineering and biotechnology research.