Researchers at The Scripps Research Institute (TSRI) claim to have created the first stable semisynthetic organism with extra bases added to its genetic code. The single-celled organism is also able to continually replicate the synthetic base pair as it divides, which could mean that future synthetic organisms may be able to carry extra genetic information in their DNA sequences indefinitely.
The cells of all organisms contain genetic information in their DNA as a two-base-pair sequence made up of four molecules – A, T, C, G (Adenine, Cytosine, Thymine, and Guanine). Each of these is known as a nucleotide (consisting of a a nitrogenous base, a phosphate molecule, and a sugar molecule) and are specifically and exclusively paired, so that only A is coupled to T and C is coupled with G. These nucleotides are connected in a chain by the covalent (electron-coupled) bonds between the sugar of one nucleotide and the phosphate of the next, which creates an alternating sugar-phosphate "backbone."
The team from TSRI have added two synthetic bases that they call "X" and "Y" into the genetic code of a E.coli carrier organism – a single-cell bacteria – and then chemically tweaked it to live, replicate, and survive with the extra DNA molecules intact.
"We've made this semisynthetic organism more life-like," said Professor Floyd Romesberg, senior author of the new study. "We can now get the light of life to stay on. That suggests that all of life's processes can be subject to manipulation."
Building on previous work on the development of X and Y in 2014, the team demonstrated at the time that engineered E. coli bacteria could hold the artificial base pair in their genetic code temporarily, but would then lose them when the organism divided.
"Your genome isn't just stable for a day," said Romesberg. "Your genome has to be stable for the scale of your lifetime. If the semisynthetic organism is going to really be an organism, it has to be able to stably maintain that information."
The addition of the X and Y base pair seemed to affect the health and well-being of the bacteria, meaning that it did not flourish, and was seen to be sluggish, and slow. To help remedy this, the team modified and improved the nucleotide transporter that ferried in the materials that allow the new base pair to be replicated.
Following this, the scientists swapped out the original Y molecule for one that was better recognized by the enzymes in DNA molecules that help out during replication, and then used the genome editing tool CRISPR to modify the bacteria's genome so that it could grow and divide normally, while still being able to pass on the X and Y nucleotides.
Working specifically on the CRISPR-Cas9 DNA segment and an enzyme that acted as part of an immune-response system where fragments of an invader genome are sampled for future responses, the team modified the genetic sequence so that it did not see X and Y as foreign bodies. As a result, the TSRI semisynthetic organism was seen to hold onto the X and Y pair in its genome even after replicating more than 60 times which, the researchers claim, means that the bacteria should be able to indefinitely hold on to the base pair.
Though only able to be used in single-cell organisms and there are no practical applications at this stage, future research is planned to work out how to transcribe the new genetic code into RNA molecules and see how they are affected when used by the cells to create proteins from DNA.
The research results were recently published in the journal Proceedings of the National Academy of Sciences.
Source: The Scripps Research Institute