In an article recently published in Science, Isaacs et al describe the replacement of all 314 TAG stop codons in the Escherichia coli genome with synonymous TAA codons, representing an unprecedented effort in large-scale genome editing.
The scientists first replaced all TAG codons in batches of ten codons across 32 separate strains using their previously-published MAGE method (Wang et al, 2009). These edited genome segments were then progressively combined using a new conjugation-based genome assembly method (CAGE). They have currently produced four strains that each have a quarter of their TAG stop codons replaced, and they hope to produce the complete TAG replacement strain in the near future. Somewhat surprisingly, no severe phenotypic consequences were observed in these replacement strains, indicating that the TAG codon is not essential, despite its near-universal presence in the genetic code of all organisms.
Indeed, the only exception to the universality of the TAG stop codon is a small selection of methanogenic archaea, and one bacterium, in which TAG encodes for the non-canonical amino acid, pyrrolysine (reviewed in Krzykci et al, 2005). Following nature’s lead, the authors hope that once they have produced the complete TAG replacement strains, they will then be able to use this free codon as a “plug-and-play” system for incorporating unnatural amino acids into proteins.
More broadly, this technology will provide an attractive alternative to wholesale chemical genome synthesis when researchers need to systematically introduce multiple genetic alterations into a genome, especially since current synthetic organism designs hew closely to natural organisms. This work may also be a first step towards creating organisms with completely rewritten genetic codes. Such fully “re-coded” organisms would have an inherent genetic “fire-wall” since they would not be able to share their genetic material via horizontal transfer or be infected by naturally occurring viruses.
Isaacs FJ, Carr PA, Wang HH, Lajoie MJ, Sterling B, Kraal L, Tolonen AC, Gianoulis TA, Goodman DB, Reppas NB, Emig CJ, Bang D, Hwang SJ, Jewett MC, Jacobson JM, Church GM (2011) Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. Science 333: 348-53
Krzycki JA (2005) The direct genetic encoding of pyrrolysine. Curr Opin Microbiol 8: 706-12
Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM (2009) Programming cells by multiplex genome engineering and accelerated evolution. Nature 460: 894-8