Why Memory?

• Just as in electronic computing and neural processing, the development of sophisticated genetic programs will require that intermediate results (or ‘system states’) and outputs are stored in cellular memories for downstream use.

• identify cell populations responsive to specific events and track their progression through the cellular response. Could have great impact on the study of any disease involving the inheritance of a cellular state, such as cancer.

• In addition to being read-outs of cellular experience, memory modules can potentially use their output as regulatory input to perform novel functions. Harnessing the ability to achieve long-term maintenance of desired relative protein levels, memory circuits might precisely regulate output or rapidly alternate between multiple outputs.

• memory modules may help overcome high production costs associated with requiring large quantities of chemical inducer.

• Deliver drugs based on the history of exposure to something eg a toxin

• Simply having another tool for storing information, retaining “state” would be very useful

The current state of the art in the field

•  transcription-based circuits: At low protein levels, the rate of expression from the gene is too low to overcome the degradation rate of the protein product, and the protein concentration is stably maintained at a low level. At high protein levels, the rate of expression is high enough to balance the degradation rate of the protein, allowing the protein concentration to be stably maintained at a high level. It was found to be sensitive to the growth rate of the cells. Transcription-based gene circuits can be knocked out of their stable state by random perturbations or unpredictable cellular influences
• recombinases flip DNA. unidirectional stable but irreversible, bidirectional is unstable and unreliable
• fine for bacteria, not great in more complex cells so far

Why the Prion Way?

Self-perpetuating. thus the synapse can be maintained after the initial trigger is gone—perhaps for a lifetime. The oligomer is extremely stable: The Orb2 oligomer is resistant to many treatments including RNase, high salt, detergents, denaturants and even boiling. Can be tightly controlled by the cell and is location and time specific- the neuronal cells this occurs in naturally have many synaptic connections but they only have oligomerization at the stimulated synapses.

- some possible ways forward

• characterised best in drosophila. Probably works similarly in mammalian cells too, proteins have mammalian equivalents…

• naturally the oligomerization happens in response to neurotransmitter tyramine (enhances Tob-Orb2 association 4x) so we could start by just adding tyramine and getting cells to record tyramine exposure. (also octopamine)

• We could also mess with levels of any of the chemicals along this pathway. could do some modelling, work out relationship between chemical concentration and level of oligomerization

• later you could get cells to produce chemicals (Lim K, Tob) in response to thing you want to sense

• the oligomer maintains memory through causing the sustained, regulated synthesis of a specific set of synaptic proteins. it does this by binding to mRNA. this can either activate or repress the translation of their target mRNAs. so we could hijack this process and get the oligomer to tell the cell to synthesise our own output proteins.

Specific things to do: sequential logic. by linking two together:

• OR gate with state associated with it and implements a circuit where the output is 1 if any of the two inputs were ever 1.

• make a delayed response and gate

Questions/stuff to investigate:

• How do you degrade the oligomer if you don’t want it anymore? So far looks like you can turn the switch on but not off.
• How modular is the oligomer protein? Could you switch the mRNA recognition domain out for another one?
• Use the switch for differentiating cells?
• Design simple experiment to see if this works in mammalian cells