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Bacterial Transformation
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.
Bacterial Transformation
Background
In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule2. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died2. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a “transformation” process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation3.
Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith’s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes4.
Bacterial Transformation using Plasmids
Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it3,5. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA and generally provide certain functional benefits, such as resistance to antibiotics6,7. In their natural environment, bacteria undergo “bacterial transformation” by taking in plasmids from other bacteria in a process called conjugation8. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.
Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create “recombinant plasmids” that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane. One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution9. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.
Applications
Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools10. In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids11. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids12. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients10. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation10. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers13. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.
References
- GL, Armstrong, LA, Conn and RW, Pinner. Trends in Infectious Disease Mortality in the United States During the 20th Century. JAMA. 1999, Vol. 281, 1 (61-66).
- F, Griffith. The Significance of Pneumococcal Types. J Hyg (Lond). . 1928 , 2 (113-59).
- Lorenz, MG and Wackernagel, F. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. . 1994 Sep;, Vol. 58, 3: (563-602).
- S, Domingues, et al. Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLOS Pathogens. 2012, 8(8): e1002837.
- Dubnau, D. DNA uptake in bacteria. Annu Rev Microbiol. . 1999, 53: (217-44).
- Solar, G del, et al. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. . 1998 , Vol. 62, 2: (434-64).
- Bennett, PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. . 2008 , Vol. 153, S1: (S347-S357).
- MLlosa, et al. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;, Vol. 45, 1: (1-8).
- Roychoudhury, A, Basu, S and Sengupta, DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian J Biochem Biophys. . 2009 , Vol. 46, 5: (395-400).
- Khan, S, et al. Role of Recombinant DNA Technology to Improve Life. I. nt J Genomics. . 2016, 2016:2405954.
- Marsischky, G and LaBaer, J. Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. Genome Res. 2004, Vol. 14, 2020-28.
- Doudna, JA and Charpentie, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014, Vol. 346, 6213-1258096.
- Good, A. Toward nitrogen-fixing plants. Science. 2018, Vol. 359, 6378: 869-70.
