Using whole cell as compartment for directed evolution of cell-DNA interaction

[Bryan Bishop]

Setup
========

Insert two nanopores into the surface of the cell membrane. Thread a
DNA molecule through the first pore and out through the second pore.

Requirements
========

DNA channel through cell membrane. A few of these already exist, I
think, such as for natural competence of certain bacteria. But also
need ways to direct a DNA molecule through a first pore and then guide
the DNA molecule towards the second pore for exit.

Also need way to ratchet cell across the length of the DNA molecule.
High utility can still be achieved even if the method of translocation
down the DNA molecule is extremely slow. These requirements are
probably the most troubling and theoretical. Once you have a mechanism
for translocation down the DNA molecule, you could tie that to
optogenetcs or something.

Implications
========

Usually there's only a DNA polymerase enzyme or other DNA binding
enzymes that get mutated. Instead, you can have the entire cell use
metabolism and all proteins directed towards optimization of whatever
you DNA-related cell properties you are selecting for.

Directed evolution of compartmentalized individual cells, rather than
individual enzymes.

Cells already keep copies of their genome available. Can replicate the
cell, or extract genomic DNA, when necessary.

Speculative utility
========

DNA sequencing: probably not. There don't seem to be any enzymes that
would be good at reading individual nucleotides that are inside of the
cell body, strung between the two pores.

DNA synthesis: again, probably not; would require a bunch of novel
enzymes, and no clear selection pressures available to encourage this?
You could look for DNA that is extruded from a cell that has a
different sequence than when it was inserted into the cell. But hard
to distinguish this from sequencing errors in the first place? Someone
would have to look at the error tolerances and measurement abilities
here.

- Bryan
http://heybryan.org/
1 512 203 0507

[Dan Bolser]

DNA is quite fragile. Have you thought about the bio mechanics?

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[Bryan Bishop]

On Mon, Nov 2, 2015 at 3:35 PM, Dan Bolser <dan.b...@gmail.com> wrote:
> DNA is quite fragile. Have you thought about the bio mechanics?

Nope, haven't thought about biomechanics. But we do see DNA moving
through cell membranes, such as with DNA translocase or ComP, ComA,
ComF, ComB, ComQ, actually here's an old diagram instead:
http://diyhpl.us/~bryan/papers2/bio/competence_proteins_ADP1.jpg
http://diyhpl.us/~bryan/papers2/bio/Internalizing_DNA.pdf.B_subtilis_competence_proteins.gif

More on "DNA uptake":
https://en.wikipedia.org/wiki/Natural_competence

[Dan Bolser]

Yup, I'm just wondering if it's strong enough to pull a cell.

[Nathan McCorkle]

I wonder if a polymerase changes color while adding nucleotides. Maybe Raman spectroscopy or something like that? Or maybe it could be coaxed to change color, so you sequence by replication. Polymerase would probably need to stay in a fixed location unless you had a multipixel camera that was really fast (faster than polymerase addition rate, available but expensive and maybe less quantum efficiency in than a sensitive single pixel detector).