In our previous installment of this series on E. coli, we explored the serendipitous discovery and adoption of this microbe as a model organism by the research community. In this installment, we will fast-forward to a more recent series of events that cemented E. coli’s place in the biotech industry.

The year was 1973, and a lot of important events where transpiring: the United States officially ended its involvement in the Vietnam War; Pink Floyd released their “Dark Side of the Moon” album, which would go on to become one of the best-selling musical albums of all time; and the first cellular telephone call was made. Few people are aware that this year also gave rise to the birth of “recombinant DNA technology” – the technological engine of the biotech industry. In November of 1973, several independent discoveries were combined in a creative way and published in a seminal scientific paper by Stanley Cohen, Annie Chang, Herbert Boyer, and Robert Helling titled, “Construction of biologically functional bacterial plasmids in vitro”1. Before diving into the main achievement of that paper, let’s lay the groundwork:

Plasmids

As early as the 1940s, scientists observed autonomous extrachromosomal DNA elements that were replicating inside certain bacteria. In 1952 Joshua Lederberg proposed the term “plasmid” to describe these extrachromosomal elements2. It wasn’t clear at the time what these plasmids were doing, but they were clearly able to replicate in the host and be inherited by daughter cells. Unlike bacterial chromosomes, which were very large (typically several million base pairs of DNA), plasmids could be quite small (some as small as just a few thousand base pairs), though we now know of the existence of plasmids that are on par with chromosomes in terms of size. At a minimum, a plasmid contains a special region of DNA that interacts with DNA replication proteins – a region commonly referred to as the “origin of replication”. Sometimes the plasmid carries the genes for its own replication proteins, while in other cases it relies on the native DNA replication machinery of the host. Additionally, plasmids usually carry genes that provide some functional benefit to their host, like genes that provide resistance to environmental stressors (such as antibiotics, heavy metals, or radiation), genes that endow the host with new metabolic capabilities (such as the ability to use new compounds as energy sources), genes that produce toxins that can kill other bacteria or predators in the local environment (thus reducing competition for the plasmid-carrying strains), or genes that allow the bacteria to colonize new environmental niches. Plasmids often also encode complex molecular machinery to facilitate their spread between hosts in the environment, ensuring their long-term propagation.

"Competent" Cells

Eventually, a method to introduce foreign DNA into E. coli was developed3. In this process, strains of E. coli that were treated with a salt solution that rendered them “competent” (i.e., able to take up DNA from their environment). When these competent cells were exposed to isolated bacteriophage DNA, the DNA was able to enter the cells where it began to function. This technique was eventually extended to plasmid DNA by Leslie Hsu and Annie Chang, enabling isolated plasmids to be introduced into new strains where they would take up residence and start replicating4.

DNA-Modifying Enzymes

In the 1960s and 1970s, two extremely important enzymes were isolated and characterized. First, in 1967, an enzyme named DNA ligase was isolated5. This enzyme, which plays a role in DNA replication and repair processes inside the cell, can take two separate fragments of DNA and chemically join them together into a single fragment. Next, in 1970 Hamilton Smith published his work on isolating and purifying a restriction endonuclease enzyme from the bacterial strain Haemophilus influenzae6–7. Restriction enzymes are so called because they were observed to “restrict” foreign DNA from functioning inside a host. Smith was able to demonstrate that these enzymes worked by recognizing a specific short sequence of DNA. If this specific short sequence of DNA was present in a longer stretch of double-stranded DNA, the restriction enzyme would bind to it, and acting like a pair of molecular scissors, cut through both strands of the DNA. These enzymes function as a kind of primitive immune system, cutting up the DNA of invading viruses and preventing infection. Following Smith’s work, a plethora of restriction enzymes were isolated from a myriad of bacterial species, each enzyme recognizing and cutting at a unique “restriction site” that is specific for that enzyme. For example, the restriction enzyme known as “EcoRI” recognizes the DNA sequence “GAATTC”.

Putting together all of the pieces

With this background information in place, we can return to November of 1973. This was the year that Cohen, Chang, Boyer, and Helling put all of these pieces together to create recombinant DNA technology.