A research group from ETH Zurich recently reported the generation of a synthetic version of the model bacterium Caulobacter crescentus genome. This work advances our knowledge on synthetic chromosome construction strategies and provides valuable insights on essential genes and regulation mechanisms.When could the synbio community claim that we have truly mastered biology? I would argue that this will be when we can de novo design and synthesize a biological system with any desired property. To get there, we need to answer two questions: what are the essential components of a living system; and how these building blocks interact with each other (and any new ones to be added).Synthesizing a genome is an intriguing way to come closer to answering these questions. It comes therefore as no surprise that some of the most celebrated and revolutionary works of synthetic biology are on building synthetic chromosomes. In 2016 the J. Craig  Venter institute reported the generation of a minimal genome for Mycoplasma mycoides, while in 2017 the Synthetic Yeast 2.0 consortium announced the redesign, synthesis, and replacement of 6 yeast chromosomes.

Caulobacter ethensis, a synthetic species

In early April, a group from ETH in Zurich added another microbe in the synthetic chromosome collection: Caulobacter ethensis-2.0.Caulobacter ethensis has a computer-generated genome, based on the Gram-negative bacterium Caulobacter crescentus. This non-pathogenic microbe is widely distributed in freshwater habitats and is a model species for cell cycle and cellular division studies.

Caulobacter crescentus. Image source: Wikimedia Commons.Jonathan Venetz and his colleagues used the C. crescentus genome as a starting point, redesigning the entire organism to produce a functional version with the smallest genome possible. Their first attempt resulted in Caulobacter ethensis-1.0, a minimal genome of around 786 kb and slightly less than 700 genes (the WT  C. crescentus is 4.02 Mb long and contains 3,767 genes). The selection of the sequences and the optimization was done computationally, mining information from previous studies where the essential genes and the respective elements are annotated.

Construction challenges

The genome of Caulobacter ethensis-1.0 was impossible to synthesize de novo. It contained several synthesis constraints, and the researchers were unable to generate the 3-4 kb building blocks that would be later assembled into the full chromosome.The scientists returned to the design board and they performed a second round of optimization: they removed the synthesis constraints—all 5,668 of them—using synonymous DNA sequences and a genome design-optimization algorithm. The derived chromosome, Caulobacter ethensis-2.0, has a radical redesign, where most putative open reading frames, ribosome stalling sequences, and stop codons have been removed.DNA synthesis of Caulobacter ethensis-2.0 was more or less straightforward. All but one of the 236 DNA blocks were easily obtained via low-cost synthesis, and the 16 megablocks were correctly assembled in yeast. The assembly of the full-length chromosome was successful and the host yeast strain maintained it intact after long cultivation (60 generations). Yet Caulobacter ethensis-2.0 does not exist as a living bacterium, as the researchers do not report the chromosome replacement of C. crescentus nor the generation of a cell that contains only the synthetic DNA.

Why this matters

Why is this work important? After all, there is no synthetic organism yet and the generated genome is not guaranteed to work as predicted in a cellular context. The answer lies in the advancement of the chromosome building know-how. Even though the genome synthesis cost has dropped and more synthesis reports become available, there are still significant challenges to face when designing a genetic sequence, let alone a full chromosome. This work shows how a combination of computational tools can accelerate the process and result in a viable synthesized mega-sequence.Probably the most important outcome from such work is the basic knowledge gained. Venetz and his fellow researchers found and corrected mis-annotated genes, tested the importance of regulatory regions, and assessed how flexible it is to insert synonymous mutations. And even though they didn’t transfer the synthetic genome into the host organism, they tested the functionality of the redesigned genes in  C. crescentus – finding that despite the radical changes, 81.5% of these genes maintained their function. This line of work shows that, while one would need to place the synthetic genome into the organism to fully validate functionality, the computational design and optimization can quicken the process. This can reduce the number of experimental iterations and test radical redesigns step-by-step.

Paving the road to synthetic cells

This all begs the question: how long until we see custom-made, fully synthetic microbes designed for particular applications? We likely will need to wait a bit longer. Even though the genetic code was decrypted decades ago, DNA seems to be more complex than just an array of digits (bases). In a crude analogy, language is not only comprised of an alphabet (DNA sequence) and grammar (cis-regulatory elements), but also syntax and expressions (DNA structural and regulatory elements).https://twitter.com/DrTomEllis/status/1113032384931422208Synthetic biology is an invaluable tool in understanding life by redesigning it, both for basic research and real-world applications. Genome synthesis is and will be a powerful tool in this process, and I am looking forward to more landmark studies. This work from Venetz et al has gained significant publicity and has been described as “impressive” by fellow scientists. It certainly has taken us one step closer to the generation of customizable cells, and I’m looking forward to further follow up studies.Article:  Venetz J, Medico L, Wölfle A, Schächle P, Bucher Y, et. al. “Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality” Proceedings of the National Academy of Sciences, 2019To learn more about emerging technologies in synthetic biology, register for SynBioBeta 2019: The Global Synthetic Biology Summit.