It’s 100 Years of E. coli!

Strains of Escherichia coli – commonly referred to as E. coli – have served as the building blocks of molecular biology for decades. In particular, K-12 was involved in numerous scientific breakthroughs.

On its 100th birthday, highlighting the contributions of E. coli to genetic engineering and biotechnology will shed light on the direction E. coli research will take in the future.

The E. coli K-12 strain was first isolated from a diphtheria patient in California in 1922. After storing the bacteria culture at Stanford University, researchers used it to teach bacterial structure in microbiology classes. The actual research potential of E. coli, however, was not explored until the early 1940s.

Codebreakers

The debut of E. coli research stemmed from questions about inheritance – prominently, how do organisms reproduce and pass on their genes? For a long time, it was thought that bacteria could only reproduce by making exact copies of themselves.

In 1946, Joshua Lederberg and Edward Tatum refuted this hypothesis by demonstrating how two E. coli bacteria could exchange genetic material through direct contact or bridge-like structures. This was regarded as the bacterial equivalent of mating.

In the following decade, E. coli was the model strain to demonstrate DNA replication elaborately. In the early 1950s, the Watson-Crick model was proposed to elucidate semi-conservative DNA replication. This mechanism was confirmed with the use of E. coli in 1957-58 by Matthew Stanley Meselson and Franklin William Stahl.

The centrifuge has also played a critical role in the research and experimentation of E. coli. Along with 100 years of this strand, it’s also the 75th year since Beckman Coulter Life Sciences launched its first centrifuge – the ‘Spinco Model E’. This unit was subsequently used in the prominent Meselson-Stahl Experiment.

In the meantime, Crick resumed his study to explore the journey extending from DNA to protein synthesis. Crick and Brenner et al discovered the triplet nature of our genetic code, which was then used by our cells to synthesise amino acids, the building blocks of proteins. Again E. coli K-12 was the model strain.

E. coli was not only central to the most beautiful experiment but also the longest-running evolution experiment. In an ongoing study, evolutionary biologist Richard Lenski has been tracking genetic mutations in 12 populations of E. coli since 1988! Throughout 70,000 generations, he observed a wide range of mutations and their impact on the phenotypes of these populations. 

One of the most striking observations was the ability of one E. coli population to grow aerobically on citrate, which is unusual for E. coli. They associated this evolution with a series of potentiating mutations, which did not affect the phenotype immediately but rather paved the way for subsequent and impactful mutations.

The long-term evolution experiment also helped us comprehend the evolution of diseases and broadened our understanding of bacteria. In my PhD. project with Patricia Foster, Professor Emerita, I made over 300 different genetic manipulations to E. coli to investigate their roles in DNA replication errors.
  
We were able to identify large regions of the E.coli genome that had higher risks of DNA replication errors. I believe that this research provides insights on how bacterial genomes are structured inside of bacteria, which were previously believed to be unstructured.

Birth of biotechnology

Considering its well-known genetics, it was inevitable that E. coli would have been at the foreground of biotechnology.

In the late 1950s, it was discovered that bacteria exchanged DNA fragments called plasmids with one another, which explained antibiotic resistance. Between 1972 and 1974, Cohen and Boyer et al. leveraged this phenomenon by inserting plasmids from one strain into the plasmid loop of another one.
  
They generated the first recombinant DNA molecule comprising DNA from two sources. Inserting the recombinant DNA into E. coli K-12, they created the first genetically modified organism, laying the foundations of synthetic biology and biotechnology.
  
E. coli was the preferred host in these experiments for many factors, including rapid growth and high efficiency of protein expression. It was also cost-effective to grow E. coli strains with a plethora of molecular tools available.

The following decades saw the power duo of E. coli and recombinant DNA technology to synthesize therapeutic proteins. The most prominent example is the production of insulin. Previously, pigs were the primary source of insulin, which made insulin expensive to obtain and inherently immunogenic. By inserting the human insulin gene into E. coli, researchers could produce large quantities of insulin, making it widely accessible to millions of diabetic patients.

Today, E. coli strains continue to be utilized for mass production of biotherapeutics. These include interleukin-2 for metastatic melanoma, human interferon-β for multiple sclerosis, certolizumab for Crohn’s disease, taxol for cancer, and blood clotting factors for haemophilia.

The natural defence mechanism of E. coli also had significant implications for gene editing. The CRISPR-Cas9 gene editing mechanism was first discovered in E. coli, where the Cas protein dissected and incorporated viral DNA fragments into its own genome to develop immunity to viral attacks. Various studies transcribed the CRISPR-Cas9 mechanism into complex organisms for gene editing, which benefited numerous applications, such as gene therapies, agriculture and biofuel production.

Lingering threat?

Inevitably, the thriving research portfolio of E. coli K-12 raised the question: “Could E. coli be dangerous?”

To answer this question, we need to distinguish between the endogenous and the lab-grown E. coli K-12. E. coli is a natural member of the human gut microbiota and is not dangerous under normal circumstances. It is an essential component of a set of strains that help us digest food rich in fiber. The ‘bad’ E. coli evolves in response to disruptions in the gut microbiome and comprises additional genes that make it pathogenic, causing gastrointestinal infections.

On the other hand, the K-12 strain and its derivatives are poor colonisers that cannot survive in the human gut. Experiments comparing the colonisation of E. coli strains found that indigenous E. coli strains outcompeted K-12 and prohibited its growth in the human gut. In addition, studies showed that E. coli K-12 could not produce the toxins that pathogenic strains produce.

Taken together, E. coli K-12 does not threaten human health as a toxic and pathogenic strain, which is yet another reason why it is an ideal candidate for biology and biotechnology.

In thE. Future

Owing to its physiological and financial advantages, E. coli K-12 is one of the most widely studied microorganisms. Approximately 30% of approved therapeutic proteins use E. coli as a host for rapid production. However, there are still opportunities for improving E. coli’s scope of biopharmaceutical production.
  
For example, E. coli does not possess post-translational modifications – like glycosylation and phosphorylation – which are crucial for protein function in humans. Preliminary studies successfully transferred glycosyltransferase genes from other bacteria and eukaryotes to express glycosyltransferases in E. coli for glycan synthesis.
  
Glycan production in E. coli has immense implications for glycoconjugate vaccine research, as several diseases from cancer to COVID-19 are associated with glycan antigens.

Furthermore, protein expression in E. coli can be optimized further to achieve better protein folding, solubility, higher yield, and ease of purification while minimising the risk of translational errors. Improving production efficiency in E. coli also requires more advanced molecular tools that produce high-throughput results and automation of synthetic biology workflows.

Despite current bottlenecks, E. coli will continue to serve pharmaceutical science and genetic engineering for many centuries to come.