Escherichia Coli: A Premier Model Organism

Introduction

Escherichia coli (E. coli) is a type of gram-negative, rod-shaped bacteria that has been used as a model organism in biology since it was discovered in 1885. The bacteria is named after German pediatrician Theodor Escherich, who discovered it in the stool of infants while looking for the cause of neonatal dysentery. E. coli has been put to good use since its discovery in 1885; discoveries involving E. coli have received eleven Nobel prizes and the bacteria has been used in countless experiments, making it one of the most studied organisms in science. E. coli became one of the most common choices as a model organism owing to the fact that it is small, reproduces quickly, and can be grown and cultured easily. These attributes and others mean that it is used from high school classrooms to premier research institutes around the world, helping provide insight to some of the biggest scientific questions of our time. As a model organism, E. coli has shaped knowledge in the fields of genetics and biology. Four groundbreaking experiments involving E. coli have been summarized below.

Experiment 1

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Figure 1. The finished table of the genetic code. "The genetic code," by OpenStax College, Biology. . [https: http://cnx.org/contents/GFy_h8cu@9.87:QEibhJMi@8/The-Genetic-Code].

Discovery of Genetic Code:

In 1961, Marshall Nirenberg and Heinrich Matthaei made the groundbreaking discovery of cracking the genetic code. While scientists had previously understood that DNA served as a template for creating RNA and proteins, the mechanism of this creation remained unknown. The question of mechanism was at the forefront of research in biology after Watson and Crick had figured out the structure of the DNA helix only a few short years before. With many researchers looking into its mechanism, Nirenberg and Matthaei were the first to figure it out. The duo did this by mimicking translation in vitro by mixing ribosomes, tRNA, and aminoacyl-tRNA synthetases from E. coli with a synthetic RNA chain of uracil bases. Essentially, they put all the tools of protein synthesis in a test tube and created their own lab-made RNA to see what proteins might be created from it. When mixed, the E. coli components read the chain of synthetic uracil bases and created a protein chain of the protein phenylalanine. The conclusion they drew was that the sequence of uracils coded for the protein phenylalanine. Having broken into one example of the genetic code, the duo then fed different sequences of RNA through the components taken from E. coli to see what proteins were created. Doing this, they found what protein each combination of RNA coded for. This experiment laid the foundation of discovering how DNA translated RNA into proteins. It allowed scientists to understand how the 64 combinations of RNA codons, based on three nucleotides each, can encode the twenty standard amino acids. These combinations make up the translation we now call ‘genetic code’. Because the proteins, such as ribosomes, could be easily harvested from the E. coli and used in vitro in the cell, E. coli once again proved itself as an effective model organism.

Experiment 2

Discovery of semi-conservative replication:

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Figure 1. Illustration of the three leading theories on DNA replication. Howell-Moroney, Madeleine, "The Meselson-Stahl Experiment". Embryo Project Encyclopedia ( 2022-01-25 ). https://hdl.handle.net/10776/13325.

After Watson and Crick proved that DNA is shaped as a double helix, the next question in the mind of many researchers was how DNA replicated. There were three possible explanations for how DNA replicated: dispersive, semi-conservative, and conservative replication. Each of these theories are illustrated in the figure to the right. The dispersive theory of replication hypothesized that the original parent strand and new daughter strand would each be broken up into several chunks and re-synthesized in a DNA strand patchwork of parent-daughter DNA. The semi-conservative theory hypothesized that one strand of parent DNA and one strand of daughter DNA would combine with each other to form the DNA double helix. The conservative replication theory said that as DNA replicated, the parent strand would remain completely intact but serve as a template for the new daughter DNA strands, creating one completely new daughter double-helix and the original parent double-helix. Meselson and Stahl used density-gradient centrifugation, a technique invented by Meselson, to settle this debate. The pair originally centrifuged DNA from a bacteriophage to try to figure out which replication theory was correct. However, the bacteriophage DNA broke apart during centrifugation and also replicated too quickly for the researchers to be able to tell which theory was correct. As a remedy, they turned to using trusty E. coli DNA.Meselson and Stahl marked the parent strand of E. coli DNA with the isotope ¹⁵N and put ¹⁴N isotopes in the mixture surrounding the DNA. These ¹⁴N isotopes could be used as part of the new nucleotides of the daughter strand. By weighing the density of the old and new DNA strands, which now varied because of the isotopes, they could see which isotopes the new daughter double-helix contained and use that knowledge to elucidate what method of DNA replication was occurring. Whirling the E. coli DNA strands around on the order of 14,000 times the force of gravity, the denser DNA was pushed towards the bottom while the lighter strands floated towards the top. The results of the density gradient centriguation showed a band with the density equal to half ¹⁴N strand DNA and half ¹⁵N isotope-labeled DNA. This band of DNA helices with a mix of the two isotopes disproved conservative DNA replication theory. Researching further, Meselson and Stalk measured the density of the new DNA strands. The density of these strands was the exact density in between ¹⁵N and ¹⁴N strands of DNA every time. If DNA followed dispersive replication theory, the density of the strands would have varied slightly between helixes. Each DNA strand being of the same density proved the semiconservative model of DNA replication. Thus, E. coli contributed to the knowledge of DNA replication because of its tightly bonded DNA and well-timed cycles of replication.

Experiment 3

Evolution in a bottle:

Over 75,000 generations of E. coli have been used in Richard Lenski’s experiment on long-term evolution. The Long Term Evolution Experiment (LTEE) is designed to explore questions about evolution in phenotype and genotype, via the model organism E. coli. The experiment began in 1988 and is still in progress today, continuing for well over twenty years. The Lenski experiment is famous in the field of biology as it is one of the most convincing pieces of evidence towards the theory of evolution. The theory of evolution has been a widely accepted theory for many years now. However, there are very few ways to collect concrete evidence towards it as in-field research is tricky, mostly due to questions regarding what defines a species, how to count whole populations, and the vast distances that many species inhabit. The Long Term Evolution Experiment has come to represent evolution in a bottle, a lab grown way of showing evolution over time. Richard Lenski began this experiment by isolating twelve identical populations of E. coli on a petri dishes, all of which came from the same ancestral source of E. coli. He fed each microbial community with a limited amount of glucose each day, putting the E. coli at a deficit in terms of resources. From this set-up, Lenski and his students watched and took samples of the populations each day, tracking their progress. With the E. coli populations reproducing up to 7 generations every day, the experiment quickly watched thousands of generations of E. coli evolve. One of the novel traits that E. coli evolved was the ability to use citrate as a source of energy. While at the beginning the populations of E. coli could not use citrate, one of the populations collected a mutation now called the Cit⁺ mutation. This mutation allowed that population of E. coli to grow and reproduce more efficiently. The Cit⁺ mutation, as well as mutations in other populations, serve as evidence that evolution occurs over time in populations. This finding provides evidence to what Darwin and the entire field of biology has long suspected: that populations can evolve. The Lenski experiment is considered a keystone in the field of modern day biology, the use of E. coli has given biologists the long looked-for evidence of evolution.

Experiment 4

Discovery and invention of CRISPR/Cas9 sequencing:

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Figure 1. External DNA being added to the DNA of a host organism. Credit: National Institute of Health https://www.nih.gov/news-events/gene-editing-digital-press-kit

In 1987 a researcher named Yoshizumi Ishini identified the first CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sequences in E. coli DNA while looking at another gene in the DNA. He had no idea that this small observation would contribute to the world of genomics being changed forever. Thanks to the recognition and application of these small DNA sequences, the world of gene editing had been unlocked. While Ishini first identified these sequences, its use as a method of genetic engineering only became apparent through the works of Jennifer Doudna and Emmanuelle Charpentier. When researching the CRISPR sequences, the duo saw that the RNA sequence stemming from CRISPR DNA, coupled with the Cas9 enzyme also found in bacteria, snipped DNA in specific spots as a way of refining DNA. Seeing this, Doudna and Charpentier used the CRISPR/Cas9 component system outside of the bacterial cell it was located in, and proved that the two-component system could be used to cut all sorts of DNA at specific places, allowing researchers to use it on DNA of their own. This technique of snipping DNA allows researchers to correct, introduce, or delete DNA sequences in a variety of organisms. The benefits of this are nearly limitless, as gene editing can be used to treat previously incurable genetic diseases such as Sickle Cell Anemia, create heartier food and crops, and even create medicines like insulin . CRISPR/Cas9 gene editing functions by using a guide RNA created from CRISPR DNA to look for a specific DNA sequence in the host genome. Once the sequence is located by the CRISPR guide RNA, the Cas9 enzyme can come in and cut through the DNA, cleaving it. Researchers can then use this precisely snipped DNA and insert their own new strands of DNA into the space where the original was cut, thus altering gene expression in the DNA. This technique of gene modification has been life changing to many sick patients, such as B-cell cancer patients, and it is thanks to research on E. coli that we even know CRISPR sequences exist.

References

Edited by [Claire Pruner], student of Joan Slonczewski for BIOL 116, 2024, Kenyon College.