Orthogonality

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In the best case scenario, the ‘minimal genome’ should be completely orthogonal: transcriptionally (SP6/T3 promoters), replicationally (Linear replication system), and translationally (16S RNA and codon usage).

The most important would be transcriptional and replicational orthogonality. Transitional orthogonality would be more difficult which is why it is less important at first. The reasoning behind this is clear: By the time the goals of a minimal-orthogonal genome are completed, it is likely that the goals of a new unnatural base pair will be as well (http://www.nature.com/nature/journal/v509/n7500/full/nature13314.html) and the unnatural base pair approach is a far superior one to conventional recode approach.

New DNA will be asked to use this expanded genetic code because of environmental reasons: it is physically impossible to spread. The minimal genome’s DNA is already in the environment because they are highly conserved essential genes.

This also brings us to the orthogonal genome and 16S RNA. (http://www.pnas.org/content/106/21/8477.full.pdf). If a modular system for genome construction is built, it would be possible to rationally swap in and out RBSs. (https://salis.psu.edu/software/)