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Three Part’s the Charm: Tripartite-chromosome E. coli Takes Center Stage

Researchers from Rikkyo University have successfully developed a tripartite-chromosome E. coli strain and formulated a new cell-free technology to synthesise gigantic DNA, revolutionising synthetic biology and synthetic genomics.

Potatoes, corn, and soy. Most of us would associate this short list of items with health foods, fresh produce, and cuisine ingredients among many other things. However, it should come as no surprise that many of these fruits and vegetables are genetically engineered to withstand harsher conditions and meet the increasing demand for food.

Genetic engineering, or genetic modification, refers to the process of directly altering the genetic makeup of organisms using recombinant DNA technology. In many cases, the DNA incorporated to target organisms consists of new, desirable traits that improve the final product. The possibilities of genetically modified organisms (GMOs) are endless, offering an array of benefits including but not limited to herbicide resistance, fortified nutrition, and higher yield.

However, to develop these GMOs, beneficial DNAs need to first be “cut” from a donor organism and cultivated in a plasmid-host before they are “pasted” into the target organisms. Escherichia coli (E. coli), a strain of bacteria with a single circular DNA consisting of about 4.6 million base pairs, is one of the most widely-used hosts for gene cloning and protein production. Not only is E. coli easily propagated in laboratory settings but it also offers high efficiency of DNA introduction into cells.

Nevertheless, there are limitations in the bacteria’s usage. Although the E. coli genome can be extracted and transferred straightforwardly, scientists still face difficulties in manipulating the DNA. Its substantial genome size, in particular, has posed a great challenge. Fortunately, a team of researchers has recently devised a simple, but equally effective solution to overcome this barrier in size.

Scientists from Rikkyo University, led by Assistant Professor Takahito Mukai and Professor Masayuki Su’etsugu, have successfully split the E. coli genome into three segments, each amounting to approximately one million base pairs. This tripartite-genome was obtained by manipulating the smallest E. coli genome strain known to date, chopping and inserting it into another E. coli bacteria.

The findings of this study not only resulted in the development of the split-genome but also demonstrated how a segmented genome can still be functional without disrupting the normal growth and proliferation of E. coli bacteria. The underlying mechanisms behind this uninterrupted process have yet to be understood, thus requiring further investigation and experimentation to uncover how replication and distribution processes are regulated in the three-part-genome.

Besides splitting the E. coli genome, the researchers have also developed a technology that deviates from the conventional cell-based DNA synthesis process. Their cell-free technique of producing gigantic DNA is a powerful amplification tool applicable to circular DNAs with up to 1 million base pairs.

Merging their findings, the team’s cell-free synthesis of split-genome could lead to the creation of artificial E. coli with valuable functions, such as material production. With this discovery, we can hope to unlock the mysteries behind genome replication and segregation, potentially advancing tools in synthetic biology and transforming ways that we manipulate genome and design life. [APBN]

Source: Yoneji et al. (2021). Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes. Nucleic Acids Research.