SYNTHSTRIPE

Synthetic gene regulatory networks for single-stripe gene expression

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 Obiettivo del progetto (Objective)

'A pivotal question in developmental biology is how cellular gene regulatory networks can respond to a signalling molecule in a concentration-dependent manner. This can now be studied using the modern tools of synthetic biology, an emerging research field that applies engineering approaches to biological systems. This project aims to adapt and develop a band-forming model in E. coli. It will explore systematically the parameter and network space that regulates the formation of a single stripe of gene expression in a morphogen gradient. The full design space of 3-gene networks, where one gene is activated by the morphogen, is 9,710 (isometric networks removed). We are therefore building a flexible network 'scaffold' that will allow the construction of any such circuit and, in particular, the six core topologies predicted in silico to form single stripes. Rational design of gene regulatory networks in vivo is extremely challenging, due to the complex interactions in living cells. Therefore, we are exploring the parameter space that leads to functional band-forming networks by building combinatorial libraries, followed by selection for an appropriate survival pressure. The synthetic networks are engineered with the potential to react as concentration band filters, with respect to a morphogen concentration. The RNA polymerases from the T7 and SP6 phages are used as activators and artificial zinc finger DNA-binding domains, cI and lacI as repressors. The output of the gene regulatory networks is a selectable gene, fused to a green fluorescent protein, which can be used for selection, counterselection and quantification.'

Introduzione (Teaser)

Who says a tiger cannot change its stripes? EU-funded researchers utilised synthetic biology to engineer stripe formation in bacteria and answer pivotal questions in developmental biology.

Descrizione progetto (Article)

Elucidating cellular gene regulatory networks (GRNs) is a complex endeavour. GRNs are naturally occurring information-processing modules that control developmental processes. To understand pattern formation in higher eukaryotes, knowing how genetic information is translated to induce cell differentiation and produce specific spatial designs is essential.

It is known that producing specific colours is dependent on morphogen concentrations where morphogen is a signalling molecule. For instance, high, middle or low concentrations of morphogen correspondingly activate a blue, white or red gene stripe. Researchers of the 'Synthetic gene regulatory networks for single-stripe gene expression' (SYNTHSTRIPE) project synthesised GRNs to engineer stripes and elucidate GRN properties and function.

In the past, researchers constructed gene circuits with predefined behaviours on a case-by-case basis. To develop three-node GRNs, project members combined modelling with experimental data on messenger RNA concentration profiles for each gene to elucidate their function. They successfully went beyond the state of the art and designed synthetic stripe-forming gene networks. More specifically, they developed a flexible network scaffold prototype with the capability to build different network topologies. In this instance, four incoherent feed-forward loop networks for stripe-forming were constructed.

Scientists used RNA polymerases from the T7 and SP6 phages as activators and selected bacterial transcription factors as repressors. Besides controlling stripe formation in these synthetic GRNs, researchers also successfully engineered an anti-stripe.

Project outcomes demonstrated that developing a unified network design space after comprehensively exploring genotype-phenotype maps is more flexible and powerful than a case-basis GRN. This synthetic biology approach could shed light on several to-date unanswered mysteries of our genome. The knowledge engendered should have wide-ranging applications ranging from biotechnology to environment protection to biomedicine.