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

1.Introduction

• Cancer is a disease that develop over time. As cells replicate their DNA, grow and divide, their genetic instability increases.
• Currenlty we are limited in the ways we are able to track cells throughout multiple rounds of cell division and growth.
• If we get better in tracking cells throughout cells division we would get valuable insights into cancer set up and progression while possibly opening new therapeutic avenues.

2.Tools people use to study Cancer are insufficient today

• 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. Mechanical, dyes, markers.

3.How to store information into living systems

• Natural systems store information, but we don't know how to act on that to control it.

From phage lambda to somatic cells, natural living systems are able to encode signals from internal and environemental imputs and store information, allowing the maintain of different cells fates, the heritability of some gene expression patterns and much more. It is indeed clear that the living medium support the encoding of information but we don't know how to control it. Th encoding information into living systems seems to be an approachable but challenging task, and we don't know yet how to do it.

• Past attempts to encode memory into living systems are technically limited"

4. Challenges associated with genetically encoded memory construction and implementation

Encoding memory will certainely not be a trivial task. The challenges associated with this project join more global questions in basic and fundamental science: Natural systems are incredibily efficient in encoding reliable behavior. However, how to implement controlled reliable behavior remains obscure.For exemples, most of the currently engineered systems have tendancy to decay with time. how can we engineer systems with reliable behavior?

To ensure success we will have a well structured approach that can be summarized as follow:

1/ We will carefully explore and choose how to write and store memory. Then we will quantitatively characterize all the parts we will use to build the memory device.

2/ We will design which device architectures are the more suited to respond to different requirements.

3/ In a similar manner, we will design which System architecture would allow the best reliability

5. Some expected outcomes of engineered memory devices

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.

II.Specific Aims

• The overall goal of our project is to establish a rationnal engineering framework supporting the engineering of a cell division counter.
• The implementation of this counter into living cells.
• The specific goal for the year covered by the fellowship to construct the first working bit of memory.

III.Research Design and Methods

Rational engineering framework

New methods for making biology easier to engineer have been proposed and applied succesfully (cite Endy 2005, Canton, 2008). These methods are in part inspirated by successfull ones issued from the fields of civil and electrical engineering. Briefly,this approach implies: decoupling complex problems into more simplers ones, using different levels of abstraction, the use of standards parts as well as a common language, and the carefull quantitative characterization of all parts and devices we will use.

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)

Counter "In digital logic and computing, a counter is a device which stores (and sometimes displays) the number of times a particular event or process has occurred, often in relationship to a clock signal. "

http://en.wikipedia.org/wiki/Counter link]

• Device architecture description

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Practical general choices

• DNA as a storage medium

We choose to store information inside DNA for the following reasons:

1/ DNA is a stable molecule. (even relatively)

2/ The natural mechanims of DNA replication allows easy transmission of the information stored inside DNA from one cell to another.

3/ Once a change in DNA sequence is done no Energy is required to maintain the state. Thus we expect this storage to be more stable in front of environemental variation.

4/ DNA modifying enzymes with exquisite specificity are found in nature. They are extensively studied, and also artificialones have been engineered.

• How to store information via DNA sequence manipulation?

A/ random: mutation (we don't want that)

B/Directed:

• • Sequence abscence or presence: Integration or excision
  • Sequence orientation: inversion
  • Sequence accessibility: DNA topology

• Using recombinases as memory writers.

We propose to use Site specific recombination as a DNA writing mechanism for the following reasons:

1/ Recombinases are efficient and very specifics for a given sequence, and each different enzyme.

2/ Lot of enzyme nature gave us, which perform a great number of different recombination reaction that we can take advantage.

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

4/ Even if they have different function they share some common catalytic domains.

5/ some attempts have succesfully changed specificity of some of them.

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Specific working lines

During the first year of the project, We will build the first bit of memory. different directions (not putting all the eggs in the same basket)

• integrase excisionase based system

Why?

The major reason is that to my knowledge,it's the only recombination system to date where we can precisely control the switch from one state to another by controlling the expression or the activity of excisionase partner. It is highly sequence specific, structure of different integrases excisionases are availables, and a lot of biochemistry.

One attractive possibility is to use the Int/Xis system to switch DNA sequences like invertases, by positionning the Att sites into the same DNA strand with an appropriate orientation, as proposed by the Boston University/ Harvard IGEM 2004 team. It is not clear however if the system could accept such different structural constraint.The 2 recombinases family use different mechanisms, and maybe one could be best suited thant the other (even if both family contain invertases).

• Engineering of enhanced Invertases.

Invertases have been used by Ham et al. and are really promising tools. they just need to be improved.

what to do?

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Concrete Experimental framework

We need to built the first unit of a memory array.

Integrase/excisionase based DNA flipping system.

*does it Flip

• • Flip flop with Lambda,phiC31,TP-901-1,HK022 integrases (screen)
  • test for reversion with Int/Xis
  • test for the ability to control genes expression via promoter switch for example(e.coli)

Conclude by selecting best working model enzymes.

A plasmid where site inversion change the restriction map.PCR assay.

• Quantitative characterization of flipping.
  • kinetics
  • improve efficiency in vitro (directed evolution, look for modular recognition domains.
  • first in vitro efficiency selection, without specific cofactors.

note: can we make a fluorescent assay?

• In vivo characterization and optimization of candidates.
  • Integrases have been shown to be able to work from bacteria to mamalian cells, and also in plants !! let's characterize if our device too!.
  • efficiency optimization in vivo: targetting the protein to the nucleus via a NLS to the proteinhas been shown to increase efficiency. Codon usage optimization.

• Integration into anhigher level system architecture.

how can we designe the memory device so that it can be scalable?

• • xploit potential modularity
  • change speificity by directed evolution

Best would be:

1 integrase catalytic domain, various DNA recognition domains, 1 Xis.

x can also measure the affinity of each enzyme for different sequences.

notes

• 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.