Researchers announced in 2011 that they had reprogrammed the genome of the bacteria E. coli so that one of DNA’s methods of encoding information went unused.  While a technological breakthrough, the scientists didn’t do anything with the new bit of genetic code.  Now only a few years later, two different groups have taken this technological tour-de-force, and are using it in the same way:  creating genetically modified organisms that may never be able to escape into the wild.

All forms of life we’re aware of use what’s called a triplet code: it takes three bases in a row in order to encode for one of the amino acids that make up a protein. A series of triplets, stretched out along the DNA, can be read to determine the precise order of amino acids. At the end of the list of amino acid codes, you’ll find what’s called a stop codon. The three stop codons (TAA, TAG, and TGA in their DNA form) don’t code for any amino acids, which the cell interprets as an indication to terminate translation of codes into amino acids.

Since there are three stop codons that mean essentially the same thing, the earlier work involved replacing all instances of one of them (TAG) with a different one (TAA). The editing process preceded in stages but, by the time it was done, all 314 cases where TAG was used as a stop codon had been replaced. This, in effect, freed up TAG to encode something else, such as an artificial amino acid.

While that sounds simple, there are a lot of things that need to be put into place before cells can start using an artificial amino acid (which may explain why these new papers are arriving over three years after the initial work). You have to either find a way to get the cells to make the artificial amino acid, or to import it from the environment. Then, you have to modify an enzyme so that the artificial amino acid gets linked to a key intermediary in protein manufacturing called a transfer RNA.

Both teams (one based at Yale, the other a Boston/Seattle collaboration) take the same approach to getting the amino acid inside a cell: they chose a large, hydrophobic molecule that can easily cross through the hydrophobic membranes that keep other molecules on the outside. They then introduced a new transfer RNA, as well as an enzyme to link the artificial amino acid to it. With that, everything was in place to get the artificial addition working as part of E. coli’s genetic code.

To reach their overall goal—making sure that the bacteria couldn’t survive outside the lab—they then had to ensure that E. coli needed this amino acid in order to survive. So, both teams obtained a list of essential proteins for which we know the full, three-dimensional structure. They then had computers search these structures for places that the artificial amino acid would fit. Once identified, the teams started going back and editing their new TAG codon into these essential genes, ensuring that they couldn’t be made without the artificial amino acid.

To an extent, this worked when just a single essential gene was modified. The bacteria grew well when they were fed the artificial amino acid, and growth quickly ground to a halt when it was taken away. But evolution is a powerful force, and about one in 106 cells would pick up a mutation that allowed it to grow further.

Some of these were mutations elsewhere in the essential protein that allowed them to tolerate amino acids that didn’t fit well. Others altered a different transfer RNA so that it replaced the one for the artificial amino acid. Still others got rid of an enzyme that normally chews up defective looking proteins. Bit by bit, the teams eliminated these potential escape routes. They also added to the number of essential genes that were modified to use the artificial amino acid.

By the time they were done, it was impossible to identify a singe bacterium that could escape its reliance on the artificial amino acid. That would mean that, even in a population of over 1012 cells, not one carries a combination of mutations that could allow them to live outside the lab conditions.

While there are differences in the details of the two papers, the end result is largely the same: an organism that can’t survive unless we feed it a very specific chemical, one that’s not normally found outside of the lab. This could be incredibly useful if we have some bacteria we’d like to keep within the lab—ones that we’re using to work with dangerous viruses or to do genetic manipulations that could have unintended consequences.

While it’s a useful tool, the public at large has long since given up on its initial worries about genetically modified bacteria. Possibly because this sort of research has gone on for decades without serious incident, labs that work with them get built all the time without a fuss. Instead, worries have focused on things like research on dangerous viruses and the use of genetically modified crops. While these new bacteria could help a bit with the former, the approach would have to be modified significantly before it could work with something as complex as a crop plant.

So, while it could be a useful tool for researchers, the development probably won’t have any affect on the public’s worries about biological research.

Image credit:  NIAID
Via Ars Technica