Organisms with alternative genetic codes resolve unassigned codons via mistranslation and ribosomal rescue.

Organisms possessing genetic codes with unassigned codons raise the question of how cellular machinery resolves such codons and how this could impact horizontal gene transfer. Here, we use a genomically recoded to examine how organisms address translation at unassigned UAG codons, which obstruct propagation of UAG-containing viruses and plasmids. Using mass spectrometry, we show that recoded organisms resolve translation at unassigned UAG codons via near-cognate suppression, dramatic frameshifting from at least -3 to +19 nucleotides, and rescue by -encoded tmRNA, ArfA, and ArfB.

Genomic Recoding Broadly Obstructs the Propagation of Horizontally Transferred Genetic Elements.

Horizontally transferred genetic elements such as viruses and conjugative plasmids move DNA between organisms, increasing genetic diversity but destabilizing engineered biological systems. Here, we used a genomically recoded Escherichia coli strain lacking UAG stop codons and the recognition protein release factor 1 to study how an alternative genetic code influences horizontally transferred genetic element propagation. The alternative genetic code conferred resistance to multiple viruses (λ, M13, P1, MS2) at titers up to 10(11) PFU/ml and impaired conjugative plasmids (F and RK2) up to 10(5)-fold.

Merlin: Computer-Aided Oligonucleotide Design for Large Scale Genome Engineering with MAGE.

Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE).

Evolution of translation machinery in recoded bacteria enables multi-site incorporation of nonstandard amino acids.

Expansion of the genetic code with nonstandard amino acids (nsAAs) has enabled biosynthesis of proteins with diverse new chemistries. However, this technology has been largely restricted to proteins containing a single or few nsAA instances. Here we describe an in vivo evolution approach in a genomically recoded Escherichia coli strain for the selection of orthogonal translation systems capable of multi-site nsAA incorporation.

A flexible codon in genomically recoded Escherichia coli permits programmable protein phosphorylation.

Biochemical investigation of protein phosphorylation events is limited by inefficient production of the phosphorylated and non-phosphorylated forms of full-length proteins. Here using a genomically recoded strain of E. coli with a flexible UAG codon we produce site-specific serine- or phosphoserine-containing proteins, with purities approaching 90%, from a single recombinant DNA.

Precise manipulation of bacterial chromosomes by conjugative assembly genome engineering.

Conjugative assembly genome engineering (CAGE) is a precise method of genome assembly using conjugation to hierarchically combine distinct genotypes from multiple Escherichia coli strains into a single chimeric genome. CAGE permits large-scale transfer of specified genomic regions between strains without constraints imposed by in vitro manipulations. Strains are assembled in a pairwise manner by establishing a donor strain that harbors conjugation machinery and a recipient strain that receives DNA from the donor.