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Generating a Library of Orthogonal Genetic Switches

RNA polymerase

Image credit: Fdardel, Wikimedia Commons, licensed under CC BY-SA 3.0

Georg Fritz is an Independent Group Leader at the LOEWE Center for Synthetic Microbiology, Philipps-University Marburg. We caught up with Georg ahead of 4Bio.

What do you think is the major limitation to synthetic biology nowadays?

As you know, synthetic biology aims to apply engineering principles to biological systems. So far however, synbio lacks simple and universally applicable rules because of the inherent complexity of living cells. This is evidenced by synthetic circuits frequently losing functionality when placed in different genetic backgrounds of the same species.

Comparative studies of synthetic circuits across different species have rarely been performed. As a result, rational forward design of synthetic circuits is greatly hampered by the limited quantitative understanding of complex biological processes even in simple bacteria. Therefore I think that orthogonality sensu stricto, while at the heart of virtually any classical engineering effort, is a major challenge in synthetic biology.

What does orthogonality actually mean and how does the field tackle the limitations you are describing?

An orthogonal system can be defined as a system that performs solely its designated function, and which is neither affected by nor affects the properties of the environment in which it is placed. There are already a number of efforts to minimise uncontrolled interactions between sets of regulators used for circuit design and the host cell, thereby increasing their degree of orthogonality.

Examples of these efforts include the development of orthogonal ribosomes for the incorporation of unnatural amino acids (“RiboX”) and RNA-based orthogonal circuitries. A particularly interesting set of transcriptional regulatory elements are so-called Extracytoplasmic Function (ECF) σ factors, which provide the working material for highly orthogonal regulatory circuits, as shown by the labs of Chris Voigt and Carol Gross.

What are ECF σ factors and how do they work?

These alternative σ factors are subunits of the RNA polymerase and are found in almost all bacterial species. They regulate diverse processes and often respond to perturbations of extracytoplasmic functions – hence their name. ECF σ factors share characteristic protein domain architectures of only two conserved regions, σ2 and σ4, which are sufficient for both promoter recognition and core RNA polymerase binding.

Today we know of more than 90 phylogenetically distinct ECF groups, which often recognise group-specific target promoter motifs. This makes them ideal building blocks for developing multiple, orthogonal switches that can be simultaneously used in a bacterial cell. Despite these advantages, ECF σ factors have so far rarely been exploited for synthetic biology applications. In the international consortium, ECFexpress, it is our goal to establish ECF σ factors as fundamental building blocks for implementing complex and orthogonal in vivo synthetic circuits.

Could you tell us more about ECFexpress?

ECFexpress was funded in 2015 in the framework of the 2nd ERASynBio joint call for transnational research projects, bringing together leading experts from the ECF field. One of our major aims is to exchange ECF σ factors between four model bacteria (Escherichia coli, Sinorhizobium meliloti, Bacillus subtilis and Streptomyces venezuelae) and thereby generate a large library of universal switches that are orthogonal across the bacterial world. However, generating libraries is definitely not enough – we also need to learn how to use these regulators!

Another aim of our consortium is to study in-depth the design rules to create truly orthogonal, ECF-based regulatory circuits. This not only involves a quantitative characterisation of ECF σ factors, but also building increasingly complex “toy circuits” which allow us to explore the boundaries of predictive circuit design.

View the abstract for Georg’s presentation in the 4Bio agenda.

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