Outline for AACR fellowship

Based on the sections they request and what we want to say.

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I. Introductory Statement, Background, and Rationale

• studying aging can give great insights into cancer biology too, as well as other fields.

• the tools people use to study aging, in particular in terms of sorting differentially aged populations are insufficient today: describe the methods

, which results (biological findings) people had using them, and comment.

• A lot of exciting discovery just waiting to be made, if we have high precision tools.

• A device allowing cells too report their own age would be great tool.

Questions: is it better to directly focus on cell counting, or to speak more generally on the usefulness of biological memory devices in general and then come to the counter? (I guess #2...)

II.Specific Aims

• we want to built a GE memory device, with a particular application to cell cycle counter.
• We will have an engineering approach, more rationale and thus more likely to deliver something that works...
• We propose as a goal for the year covered by the fellowship to construct the first working bit of memory.

Question:Do we have to take into account the fact that the fellowship begin on July, so that we'll have already progressed, and in this case we should plan a "development schedule" and see where we aim to be at this date.

III.Research Design and Methods

Introduction

• We will use different levels of abstraction (cite references nat biotech?)
• quantitative characterization of all parts and devices.
• We have already specified some performance requirements and chosen some working direction that we think has the best potential of success.
• specific concerns are:
  • Modularity
  • reuseability
  • scalability

Our design will be done following these requirements.

System architecture description

Describe the cell cycle counter in abstract (coupled gated oscillators)

Device architecture description

how the first bit should be designed?

Question: in our case do we need to focus on the particular case of a same imput switching from any state to the other?

We choose recombinases

• Why?

1/ writing DNA is interesting in terms of specificity, stability, scalability (develop this).

2/we already have a bunch of enzyme nature gave us, which perform a great number of different recombination reaction that we can take advantage.( so why don't you use kinases?)

3/well studied (lambda...), some of them already used in engineering memory (Ham); cite IGEM 2004 project also.

4/ seems that we can modify specificity and that to some extend there's a kind of modularity we could exploit (develop this precisely).

project, methods

• During the first year of the project, We will build the first bit of memory

• Encoding into DNA sequence

• We will have different strategies to ensure succes.

different directions (not putting all the eggs in the same basket)

Question: how many propositions to work on on parallel? 2, maybe maximum 3?

describe precisely what kind of DNA manipulations we' ll perform to write memory.

• we will use DNA sequences inversion

• • Integrases/excisionase

potentially flip DNA? can also be used without flipping (and is efficient!)

• Directed evolution for enhanced Invertases

• Can we use "directed DNA transposition"? I am afraid we still lack specificity but this could also be optimized and some work has already been done. ( add ref)

controlled retrotransposition via an RNA intermediate could also be usefull for combining lineage and counting...see latter for this.

and need to read more

descibed which problems can happen and what you'll do.