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BITS2007 Meeting
BITS2007 Meeting

26-28 April 2007 Napoli, Italy

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Synthetic "switches": a new way to tackle complex diseases and biotechnological innovation
This project is aimed at engineering a synthetic network for in vivo delivery of
mRNA/protein. The final intent will be to create a stable synthetic "oscillatory"
network in a mammalian system, which is able to express mRNA/protein levels with a
pre-determined frequency and amplitude. The rhythm of delivery should be
corresponding to the normal nutrient uptake. 

There are many problems to overcome when trying to build artificial networks in
living cells; inefficient inducibility, instability, stochastic effects and
background activity (leakiness) have been highlighted by scientists as the most
important ones. Previous studies indicate as design guidelines the use of strong
promoters and efficient ribosome binding sites, as well as making sure there are
tight transcriptional repression and a comparative protein and m-RNA decay. To take
into account all the possible variables it is necessary to perform a systematic
examination of the effects of parameter variation with quantitative modeling and
analysis to evaluate ranges of parameters for the experiments and to predict possible
out-comes. In this way several synthetic networks constructed by rearranging
regulatory components in a cell have been characterized. In this project we will
follow the experimental method described by Gardener et al. [1] and Kramer et al.
[2]. These two groups used first a mathematical model to design a toggle to be
implemented respectively in E. Coli and Chinese hamster ovary cells and then built
the synthetic toggle in vivo. We are planning to build a switch based on the toggle
designed by Collins and his group, in which each promoter is inhibited by the
repressor that is transcribed by the opposing promoter; in the absence of inducers,
two stable states are possible: 

A.	promoter 1 transcribes repressor 2
B.	promoter 2 transcribes repressor 1

The switch is obtained by introduction of an external inducer of the currently active
repressor (1 or 2). The inducer permits the opposing repressor to be maximally
transcribed until it stably represses the original active promoter. The novelty is
that we will use shRNA to express either a siRNA to silence transcription by
increasing degradation level of mRNA [3], or to express a microRNA to block
translation of the protein [3]. In both cases, the shRNA will act as a repressor so
that, having a control at the level of transcription/translation should avoid
problems with leakiness of the promoters, as the proteins produced would be
"silenced". The use of lentiviral vectors throughout the study will allow testing the
circuit on primary cell and in animal models. Specifically, once built, the toggle
will be used to produce a therapeutic protein in animal model of genetic diseases to
test the possibility of inducing the protein in vivo only when needed. In this latter
system, individual cellular oscillators will be synchronized to fulfill the
macroscopic function of a protein delivery device.

The final goal of the project is to use synthetic inducible switches for therapeutic
Id: 113
Place: Napoli, Italy
Centro Congressi "Federico II"
Via Partenope 36
Starting date:
-- not yet scheduled --   
Duration: 01h00'
Contribution type: Poster
Primary Authors: CUCCATO, Giulia (Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111 80131, Napoli, Italy)
Co-Authors: MARUCCI, Lucia (Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111 80131, Napoli, Italy)
VELIA, Siciliano (Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111 80131, Napoli, Italy)
DI BERNARDO, Diego (Telethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111 80131, Napoli, ItalyTelethon Institute of Genetics and Medicine (TIGEM), Via P. Castellino 111 80131, Napoli, Italy)
Presenters: CUCCATO, Giulia
Included in session: Poster Session
Included in track: Gene expression and system biology | Last modified 08 July 2009 10:35 |

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