Splitting the Escherichia coli genome into three parts to create new cells
- Genome installation technology for model organisms -



A research group led by Assistant Prof. Takahito Mukai and Prof. Masayuki Su’etsugu of the College of Science, Rikkyo University, has developed a technique for splitting and installing Escherichia coli genomes. Their research was conducted as part of the JST Strategic Basic Research Programs.

The E. coli genome consists of a single circular DNA with 4.6 million base pairs. Conventionally, this has been too large to be extracted from an E. coli cell for manipulation and transplantation into another E. coli cell. In their study, the group succeeded in reducing the size of the E. coli genome by splitting it into three parts (each with 1 million base pairs) and developed a technique for extracting the split genome from an E. coli cell and installing it in another cell. Their findings could be used not only to better understand the mechanism of genome replication and partitioning, but also as a synthetic biology tool for replacing genomes—the blueprints of life—to create functionally designed organisms.

The results of their research were published online as a Breakthrough Article in the British scientific journal "Nucleic Acids Research" on April 28, 2021 (UK time).

Main points

  • The researchers maintained an E. coli genome as 3 pieces of circular DNA consisting of 1 million base pairs each.
  • They developed a technique for extracting the split genome from an E. coli cell and transplanting it into another cell.
  • Future applications of this technique are anticipated in synthetic biology, such as for transplanting an artificially synthesized split genome or constructing artificial E. coli designed to have useful functions.
The findings were obtained through the following projects, research fields, and research topics.

JST Strategic Basic Research Programs team research (Core Research for Evolutional Science and Technology: CREST)
  • Research field: "Creation of cell control technology using genome-scale DNA design and synthesis" (Research supervisor: Prof. Haruhiko Shiomi, Keio University School of Medicine)
  • Research topic: "Cell-free on-chip synthesis and activation of artificial genomes"
  • Principal Investigator: Masayuki Su’etsugu (Professor, Rikkyo University College of Science)
  • Research period: October 2018 - March 2023

In this study, the group developed a cell-free on-chip method of genome synthesis that uses in vitro reconstruction techniques and microfabrication/manipulation techniques. They evaluated the process of transplanting a synthetic genome into cells and booting-up it, with the objective of developing a high-speed, low-cost cycle of "genome-synthesis, booting-up, evaluation." Using the genome synthesis techniques they developed, they plan to proceed with minimizing and defragmenting the E. coli genome to construct a "minimum platform cell" that simplifies the genome to a level that is completely understandable.

Study background and process

The genome is the blueprint for life and can be thought of as an operating system encoded by four-letter DNA sequences. The hardware for booting the operating system is a cell enclosed in a membrane. Recent advances in synthetic biology have made it possible to rewrite these operating systems to artificially design cell functions. A prime example of this is a study from the J. Craig Venter Institute from 2010. In this study, researchers reported creating a new cell by artificially synthesizing the entire genome (1 million base pairs) of a small mycoplasma bacterium and installing it in another cell.

However, the organisms with genomes that can be installed with this method are still limited to mycoplasma, and there have been no successes with model organisms that are widely used in research and industry, such as E. coli. One challenge is the genome size. The E. coli genome, at 4.6 million base pairs, is more than four times larger than the mycoplasma genome, and its physical length is 2 millimeters, which is nearly 1,000 times the size of an E. coli cell. Although it is compactly folded inside the cell, it becomes severed and broken as soon as it is taken outside.

This group investigated ways of splitting the E. coli genome and maintaining it as multiple small chromosomes as a means of achieving genome installation. In E. coli and many other bacteria, the genome is encoded by a single large circular chromosome, though in eukaryotes this is encoded and divided into multiple chromosomes. If the chromosomes could be divided into pieces similar to mycoplasma size, the split chromosomes could be taken in and out of E. coli cells and installed in other cells to create artificial E. coli. This is what the group envisioned as they set out on their research.

Content of the study

The group used E. coli minimum genome strain which was constructed by removing any unnecessary genes. The size of the minimum genome is 3 million base pairs. Employing a technique for retrieving large sections of the genome inside E. coli cells using a site-specific recombination mechanism, this minimum genome was made even smaller by dividing it into three sections of 1 million base pairs each (Figure 1). For an E. coli cell with a genome divided into three parts to survive, the extracted region must be stably replicated and partitioned inside the cell as split chromosomes. Therefore, they investigated the replication origin (ori) and the partitioning system (par), which are essential for maintaining the split chromosomes. They found that an ori-par system in the secondary chromosome of a Vibiro bacterium1 and a sop partitioning system derived from a giant plasmid1 were effective for stabilizing the split chromosomes. The chromosome split in this way was stably maintained for more than 100 generations its three-part state: a chromosome consisting of a genomic region containing the original oriC (Figure 1 center, gray) and chromosomes that were maintained by the Vibiro-derived ori-par system and the oriC-sop system (Figure 1 center, orange and green). The cell growth rate of the E. coli strain was not significantly inhibited.

Next, they investigated a method for extracting and purifying the three split chromosomes with 1 million base pairs each from the E. coli as supercoiled DNA2. Surprisingly, the purified split chromosome could be introduced into a different E. coli strain with natural chromosomes consisting of 4.6 million base pairs using electroporation, a classical method of creating pores in a cell membrane using electrical pulses to introduce DNA (Figure 1). This is significantly larger than the DNA that had been previously introduced into an E. coli cell. In addition, they showed it is possible to exchange split chromosomes between E. coli strains that have three split chromosomes.

Future prospects

This ground-breaking study demonstrated that stable E. coli growth is possible even if the genome is split into three parts. Figuring out how to control the replication and partitioning of the three split chromosomes is an important task going forward.

In this study, researchers for the first time succeeded in extracting genome-sized DNA with 1 million base pairs from E. coli and installing it in another cell. The group is also developing a technique for synthesizing large DNA outside of cells (cell-free) and has reported on a technique for cell-free amplification of circular DNA with 1 million base pairs. In the future, the ability to install split chromosomes synthesized using cell-free methods into E. coli cells could help create artificial E. coli designed to have useful functions, such as the ability to produce certain substances.


Figure 1. Creation of a three-part Escherichia coli genome and installation of the split genome

The size of a natural E. coli genome is 4.6 megabase (4.6 million base pairs) and is difficult to extract from and manipulate outside the cell. Therefore, the genome was reduced in size to 1 megabase (1 million base pairs) by splitting it into three parts. This split genome could be removed from an E. coli cell and installed into another cell.


1) ori-par system, sop partitioning system
The ori-par system consists of a DNA region (ori) that encodes the replication initiation sequence and a DNA region (par) that partitions the DNA after replication. Together they stably maintain large DNA in bacteria that proliferate via cell division. The sop partitioning system is a par system derived from the E. coli F plasmid.

2) Circular supercoil DNA
A form of circular double-stranded DNA (closed-ring DNA) twisted into a compact state.


“Grand scale genome manipulation via chromosome swapping in Escherichia coli programmed by three one megabase chromosomes”

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