Orthogonal Replication


Orthogonal replication is a pretty large issue when it comes to directed evolution. Directed evolution requires that one DNA fragment gets mutated quicker than others. This is usually achieved in vitro, but there are advances in making this a cellular in vivo environment. However, this can also be used to segragate a cell to have 2 independent systems: an orthogonal user defined system, and a normal cell replication system.

The main point behind this is separation of systems. For example, computer systems such as Freebsd separate kernel userland and base system. In biology, this idea could be useful for creating a replicating cell and user defined systems to run on top of it.

In essence, there would be 2 important parts. The base system/kernel (that comes with the strain) and then whatever a person may desire to clone. The kernel base system would include a normal functional translation system, untouched (excluding TAG codons) that’s job is simply to replicate the cell. It would hold ribosome genes, tRNAs, replication genes, and essential biosynthesis genes. This would be at a low copy number. All systems it uses would stay intact in their original form until more modification could be done.

Next would be the base system. the base system essentially makes a strain a strain. It can hold genes for biosynthesis of molecules to grow faster, for biosynthesis of target molecules, for extra replication proteins and tRNA pairs. It is recommended that the base system be created with orthogonal 16S RNA RBS (paper) and an orthogonal transcription system (self regulating T& complex.. This would be on a low copy number plasmid.

Finally, there would be the userland. The userland would be anything else that gets put in. This could be ANYTHING the user chooses to define. Orthogonality would be strongly recommended for this plasmid. Complete orthogonality, with a different transcription system, a different translation system (unnatural base pairs and tRNAs for them) different 16S RNA RBSs, different replication system. This goes for defining a completely new outlook on synthetic biology.

In the goals of making a modular genome, why orthogonality? The key is in separation of systems. Yeast are extremely good at this: they have 2 translation systems, 3 different locations for replication (cytoplasma, nucleus, mitochondria) and a possible 3 different transcription systems (pGKL1/2, nucleus, mitochondria). Bacteria aren’t. E coli only has 1 location for all of these, the inside of its cell. So, you may ask, why do we use it? Because it is the simplest system there is- the main goal of the entire project is not the necessarily make a *minimal system* but a *simple system*. Simple is best for understanding, something that needs to be