Genome engineering used to create a bacterial kill switch

Spencer Katz

In 2011, researchers announced that they had reprogrammed the genome of the bacteria E. coli, changing it so that one of DNA's methods of encoding information went unused. While a technological tour-de-force, the scientists didn't actually do anything with the newly available bit of genetic code. Now a few years later, two different groups have used it to accomplish the same end: 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 goalmaking sure that the bacteria couldn't survive outside the labthey 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.

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Genome engineering used to create a bacterial kill switch