Adding letters to the alphabet: scientists create the first living organism with semi-synthetic genetic code

There have been some really interesting reports in genomics and DNA technology this week!  One in particular, has found a way to expand the DNA "alphabet" of Escherichia coli.

DNA is made up of four nucleotides, each containing a nitrogenous base, a five-carbon sugar backbone (either ribose or deoxyribose), and at least one phosphate group.  The nucleotides form hydrogen bonds with each other, forming base pairs, specific to their hydrogen bonding capacity.  Adenine (A) binds with Thymine (T), and Cytosine (C) binds with Guanine (G).  This is the basis for every single DNA strand we have ever encountered in any living organism on the planet.  All of your genetic information is encoded by these four nucleotides.

A group out of The Scripps Research Institute in California created an additional pair of unnatural (not found in nature) nucleotides, and expressed them stably in E. coli.  That's actually incredibly complicated, because the unnatural nucleotides have to be present in the cell in a concentration that allows them to be integrated into DNA strands, they have to line up alongside the natural nucleotides so as to not interfere with the structure of the DNA strand, and they have to be recognized by the cell's internal DNA replication machinery.  This is what the two unnatural base pairs (UBP) look like, compared to the the G-C bonding at the bottom of that figure:

The authors describe several ideas they had to ensure adequate amounts of UBP in the cells, but ultimately decided to focus on nucleotide triphosphate transporters (NTT), which transport the substrates, or building blocks, of nucleotides across membranes.  Successful transport across the membranes required that the UBP in the growth medium be stable.  Using High Performance Liquid Chromatography (HPLC), were able to quantify the uptake of the UBP into the cells.  The authors also validated that the UBP could be replicated using different DNA polymerases in vitro, finding that DNA polymerase I was suitable for replication.  But since E. coli typically use DNA polymerase III to replicate their genome, the authors engineered a plasmid to focus replication of UBP using polymerase I.  Just to give you an idea of the scope of this project, so far all of this is only the preliminary part of this paper!  I've never seen a paper with such a long methods section, I'm awed by this work.

The researchers then transformed the E. coli cells to express high amounts of NTT, and grew them in media containing the UBP.  After 15 hours, they looked to see how well the plasmids containing the UBP were replicated - meaning that they verified that the cells not only incorporated the UBP in their genome, but copied the DNA that contained them, with a fidelity of 99.4%!  That means that the unnatural nucleotides are not being removed from the genome through the cell's own DNA repair mechanisms - the only way that the UBP were removed from the genome was when they were not being supplied in the growth medium (which means that if these cells were to escape the lab, the UBP would be unlikely to cause any damage to the natural world).  This technology could eventually be applied to create a platform for synthetic biology with a range of applications, from site-specific labeling of nucleic acids in living cells, to the production and evolution of synthetic proteins with unnatural amino acids.

In fact, I am most interested in future studies of this system that look beyond DNA replication to gene expression!  I'm so curious to find out if and how these cells will translate these unnatural nucleotides into amino acids and how the resulting protein will look and function.