ST-FLOW

Standarization and orthogonalization of the gene expression flow for robust engineering of NTN (new-to-nature) biological properties

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 Obiettivo del progetto (Objective)

'The ST-FLOW Project merges the efforts of 14 leading European research groups for developing material and computational standards that enable the forward-design of prokaryotic systems with a degree of robustness and predictability that is not possible with customary Genetic Engineering. The central issue at stake is the identification and implementation of rules that allow the conversion of given biological parts assembled with a set of principles for physical composition into perfectly predictable functional properties of the resulting devices, modules and entire systems. ST-FLOW focuses on each of the steps that go from assembling a DNA sequence encoding all necessary expression signals in a prokaryotic host (by default, E. coli) all the way to the making of the final product or to the behaviour of single cells and populations. Two complementary approaches will be adopted to solve the conundrum of physical composition vs. biological functionality of thereby engineered devices. In one case (bottom up), large combinatorial libraries of gene expression signals will be merged with suitable reporter systems and the input/output functions examined and parameterized in a high-throughput fashion. The expected outcome of this effort is to establish experience-based but still reliable rules and criteria for the assembly of new devices and systems -following the same physical composition rules or adopting CAD design. Yet, many outliers (combinations that do not follow the rules) are expected, and making sense of them will be the task of the complementary top-down approach. In this case, ST-FLOW will revisit some of gaps in our knowledge of the gene expression flow (transcription, mRNA fate, translation) that need to be addressed for engineering functional devices from first principles. Ethical, legal and societal issues will also be examined in a context of public dialogue and sound science communication.'

Introduzione (Teaser)

From food to fuel, bacteria are routinely used in various applications. To widen their range of industrial applications, European scientists developed tools and standards for obtaining genetically engineered bacterial clones.

Descrizione progetto (Article)

Bacteria are routinely used in biotechnological and industrial applications that exploit their natural chemical reactions. Additionally, with the advent of genetic engineering they can be programmed to produce substances that could be utilised in food manufacturing, agriculture and medicine.

Escherichia coli is the most commonly used bacterial strain in molecular biology. Attempts to expand the engineering toolbox beyond E. coli have been met with difficulty and were largely application-dependent. To be able to assemble specific DNA segments together in a high-throughput manner and design synthetic genomes, better molecular tools are required.

Using a combination of synthetic biology, engineering and bioinformatics, the EU-funded http://www.cnb.csic.es/~stflow-project/ST-Flow/Welcome.html (ST-FLOW) project set out to develop molecular tools and computational standards for reproducibly obtaining genetically engineered bacteria.

The consortium wished to build on existing knowledge of biochemical processes and organisms, and improve the industrial exploitability of the biological world.

For this purpose, they optimised all the necessary steps from DNA sequence design to construction of biosynthetic pathways or interacting systems that respond to external signals. A DNA assembly strategy called Modular Overlap-Directed Assembly with Linkers (MODAL) was established for putting together compatible pieces of DNA.

The physical assembly of DNA functional modules (origins of replication, antibiotic markers, expression systems and reporter genes) led to the creation of an online database of plasmid and transposon vectors. These molecular tools were designed with potential applicability for both gram-negative and gram-positive bacteria.

Predicting the behaviour of a synthetic genetic system requires detailed knowledge of its component parts. As a result, ST-FLOW partners standardised the methods for determining the functional relationship between a transcriptional regulator and its target promoter. Additionally, they performed a transposon mutagenesis study that enabled them to identify genomic regions with sustained transcriptional capacity. Vector constructs containing sensor elements that could respond to external or internal signals were also designed.

The ST-FLOW platform is expected to find immediate application in the industrial manufacture of bacterial strains tailored for biocatalysis speeding up chemical reactions in production. The new bacteria can also be used as biosensors to detectenvironmental pollutants.