Researchers from the United States, China and France have created what they say is the world’s first stable semi-synthetic microorganism. The research appears in the Proceedings of the National Academy of Sciences.
Life’s genetic code has only ever contained four natural bases: adenine (A), cytosine (C), guanine (G) and thymine (T).
These bases pair up to form two ‘base pairs’ — the rungs of the DNA ladder — and they have simply been rearranged to create bacteria and butterflies, penguins and people.
Building on their 2014 study in which they synthesized a DNA base pair, Scripps Research Institute Professor Floyd Romesberg and co-authors engineered the bacterium Escherichia coli that uses the four natural bases, but that also holds as a pair two synthetic bases (X and Y) in its genetic code.
“The genetic alphabet encodes all biological information, but it is limited to four letters that form two base pairs,” the authors explained.
“To expand the alphabet, we developed synthetic nucleotides that pair to form an unnatural base pair (UBP), and used it as the basis of a semi-synthetic organism (SSO) that stores increased information.”
“However, the SSO grew poorly and lost the UBP under a variety of standard growth conditions.”
The researchers have now shown that their bacterium can hold on indefinitely to the synthetic base pair as it divides.
“We’ve made this semi-synthetic organism more life-like,” Prof. Romesberg noted.
“While applications for this kind of organism are still far in the future, this work could be used to create new functions for single-celled organisms that play important roles in drug discovery and much more.”
In the study, Prof. Romesberg and co-authors developed the means for the single-celled organism to retain the artificial base pair.
First, they optimized a tool called a nucleotide transporter, which brings the materials necessary for the unnatural base pair to be copied across the cell membrane.
“The transporter was used in the 2014 study, but it made the semi-synthetic organism very sick,” said study first author Yorke Zhang, a graduate student at the Scripps Research Institute.
“We discovered a modification to the transporter that alleviated this problem, making it much easier for the organism to grow and divide while holding on to X and Y.”
Next, the researchers optimized their previous version of Y.
The new Y was a chemically different molecule that could be better recognized by the enzymes that synthesize DNA molecules during DNA replication. This made it easier for cells to copy the synthetic base pair.
Finally, they set up a ‘spell check’ system for the organism using CRISPR-Cas9, an increasingly popular tool in human genome editing experiments.
But instead of editing a genome, they took advantage of CRISPR-Cas9’s original role in bacteria.
The scientists designed their Escherichia coli to see a genetic sequence without X and Y as a foreign invader.
A cell that dropped X and Y would be marked for destruction, leaving the team with an organism that could hold on to the new bases. It was like the organism was immune to unnatural base pair loss.
“We were able to address the problem at a fundamental level,” said co-author Dr. Brian Lamb, of Vertex Pharmaceuticals.
The team’s semi-synthetic bacterium was thus able to keep X and Y in its genome after dividing 60 times, leading the researchers to believe it can hold on to the base pair indefinitely.
“We can now get the light of life to stay on. That suggests that all of life’s processes can be subject to manipulation,” Prof. Romesberg said.
Yorke Zhang et al. A semisynthetic organism engineered for the stable expansion of the genetic alphabet. PNAS, published online January 23, 2017; doi: 10.1073/pnas.1616443114
This article is based on a press-release from the Scripps Research Institute.