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, associated with abnormal cell proliferation and differentiation. Thus, being able to track proliferating cells is important to understand cancer progression, for example isolating highly proliferative calls on a population subset. Diagnosis, Therapy with stems cells, track stem cells proliferation. follow proliferation of cells implants, following Cancer stem cells proliferation...
 * 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 diagnostostic and therapeutic avenues.

2.Tools people use to study Cancer are insufficient today

, which results (biological findings) people had using them, and comment. Mechanical, dyes, markers.
 * the tools people use to study cell proliferation, in particular in terms of sorting differentially aged populations are insufficient today: describe the methods

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
1/introduction

a/ Establishment of an engineering framework for the engineering of cell cycle counter


 * background
 * lacks of standards in bioengineering lead to slow progress and poor reusability.
 * 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
 * extensive use of DNA synthesis will speed up the work.

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. ''
 * performance requirements
 * We have already specified some performance requirements
 * specific concerns are:reliability, modularity, reuseability, scalability, portability, reactivity
 * Our choices will be done trying to meet requirements in the best way possible.

b/ Proposed architecture for a cell cycle counter


 * System architecture description
 * System: counter
 * coupled single bit memory devices
 * ripple counter

''notes: A 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."In our project, the overall biological counter is considered as the system. Different parts, proteins, promoters, repressors, are combined together to built a memory storing device, which is able to switch between 2 different states in response to an external stimulus (or input) and to display its current state to the experimentator (output). In this system, each device is able toof store to one bit of information, by analogy with digital bits. Thus the counter capacity is direclty linked to the number of bits/devices and will be able to store 2n states where n is the number of devices. In the case of a cell cycle counter, each bit correspond to a round of cell division.''

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


 * Device architecture description
 * single bit memory device made of sub-devices.
 * sub devices are a memory writer and a state display device.
 * each subdevices are composed of parts, DNA sequences or proteins enzymes, regulatory domains etc...

''As a practical example, for coupling writing events to cell cycle, one part could be a cell cycle dependent degradation tag.The counter will be composed of multiple memory storing devices, each able to store one bit of memory. Each memory storage device will be composed of subdevices made different parts: a "memory writing" device, a State display device...''

put a a figure

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.

Specific working directions
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).

Preliminary work by mizuuchi (jmolbiol, 1980) and nash and pollock 1983; lambda integrase swith, change in dna likning number. Cite crelox systems also invert sequence, same family ; Hoess, NAR, 1986, lot of others, and Kano BBRC 1998. Calos lab shown that PhiC31, TP901-1 and R4 integrase are able to perform deletion when sites are in a specific orientation on a same chromosome, so potential inversion too can also measure the affinity of each enzyme for different sequences. notes


 * '''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?

Concrete Experimental framework
1/ Integrase/excisionase based DNA flipping system.


 * screening for different int/Xs pair ability to flip a synthetic target DNA sequence
 * 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 the first round of selection selecting best working model enzymes candidates. 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) but first if works in vivo.
 * first in vitro efficiency selection, without specific cofactors (portability).

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. example: targetting the protein to the nucleus via a NLS to the protein has been shown to increase efficiency. Codon usage optimization.


 * Integration into an higher level system architecture.
 * our concern: make something scalable and reusable in different contexts.
 * exploit potential modularity
 * change specificity by directed evolution

Best would be:

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

Make a figure.

=notes=

In vivo and in vitro optimization hands in hands...