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Synthetic Biology in the Freemont lab

Synthetic biology is an application-driven field attempting to apply fundamental principles of engineering to the redesign of biological systems, to produce valuable and novel biological functions.  The driving force behind the field is the desire to develop robust biological systems rapidly and efficiently.  Potential applications of synthetic biology are numerous and range from the production of bio-fuel to the synthesis of biomaterials. For a general overview of the design principles and tools for synthetic biology see MacDonald et al., 2011 and Kelwick et al., 2014. The lab also maintains an online community to track the latest developments in the field - the Synthetic Biology Index of Tools and Software (SynBITS).

The engineering framework that the lab has adopted is shown in Figure 1.  The idea is that through multiple reiterations of this cycle, a biological system can be refined.  In addition to application-driven projects, the lab is interested in the expansion of foundational technologies to enhance the workflow through the cycle, for example the expansion of software for the design and modelling of biological systems.

engineering framework behind synthetic biology

Figure 1: The engineering design cycle in synthetic biology.

Since 2006 the lab has helped to assist and advice undergraduate students to develop a synthetic biology-based project for the iGEM (International Genetically Engineered Machine, see sidebar) competition.  This competition is based around mixed disciplines undergraduate students collaborating in the design of a synthetic biology project, the end product of which they present at the iGEM jamboree at MIT.

The lab is part of a collaborative effort concentrated in the Centre for Synthetic Biology and Innovation at the Imperial College Institute of Systems and Synthetic Biology. Our external collaborators include the Flowers consortium, the Synthetic Yeast 2.0 project (UK SUGER) and the Syntegron consortium. The Flowers consortium is involved in creating a UK infrastructure for synthetic biology.

We carry out research in a number of areas including cell-free systems, biosensors, the Synthetic Yeast project, small molecule biosynthesis and computational protein design.

Cell-free biofilm biosensor

Team: Ke Yan Wen

Collaborators: Prof. Alain Filloux and Prof. Jane Davies

Cell-free synthetic biology has gained increased popularity in recent years as an alternative platform to live cells, enabling the study of biological systems in an environment of reduced complexity. We have developed biosensors in cell-free systems for the detection of pathogenic bacteria, with a focus on Pseudomonas aeruginosa – an opportunistic pathogen that can cause persistent infections in humans with compromised immune systems. It is a particular concern for cystic fibrosis patients, as it is able to colonise the lung and is resistant to antibiotic treatment.

To detect the presence of these bacteria, we utilised their natural cell-to-cell communication mechanism, known as quorum sensing (QS). Biosensors have been constructed using the native response elements of bacteria and coupled to a detectable output. This enables the detection of QS signalling molecules produced by bacteria, while its implementation in a cell-free platform provides improved biosafety when compared to whole-cell systems. After demonstrating that these biosensors are able to detect endogenous signalling molecules from cultured P. aeruginosa, we have now progressed to testing clinical samples from cystic fibrosis patients with P.aeruginosa infections. Ultimately the aim is to provide an improved detection method that can aid in the diagnosis and monitoring of bacterial infections.

Parasight - Schistosoma parasite biosensor

Team: Dr. Alexander J. Webb

Collaborators:

One of the primary goals of synthetic biology is the application-driven generation of new parts, circuits, and systems to solve problems that as yet have not been adequately addressed. Parasitic diseases affect millions of people worldwide, causing a range of illnesses and death. Rapid and cheap ways to detect parasites at the sites where infections occur are needed to break the disease cycle. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages of their life cycles. We propose to use synthetic biology to build an affordable and molecular cell-based biosensor as a generic platform that can be applied to quickly and easily detect parasites via their protease signatures.

The parasitic infection Schistosomiasis affects over 200 million people worldwide, with estimates suggesting that a further 780 million people are at risk of infection. The causative agents are fluke worms of the Schistosoma genus, and infection only occurs when the cercarial larvae are able to penetrate the skin. To facilitate this, the cercariae secrete an elastase possessing defined substrate specificity. Our project takes advantage of this property of the cercarial elastase, and has used it to design and create biosensors that are specific in targeting Schistosoma. The design of our biosensors is based on a two-pronged approach, 1) an accurate detection system which is targeted to the cercarial elastase, and 2) an easily measurable output. Using synthetic biology approaches we have engineered the biosensors to be “housed” in two bacterial chassis, Bacillus subtilis and Escherichia coli, and we have further designs that will enable for a cell-free based biosensor.

Optimisation of cell-free transcription and translation systems

Team: Dr. James T. MacDonald and Dr. Richard Kelwick

Collaborators:

Cell-free biological systems allow the in vitro transcription and translation of genetic circuits by extracting the transcription and translation cellular machinery from cells and combining this with a DNA template, an energy solution containing various cofactors, amino acids and a secondary energy source. Most typically these systems are derived from E. coli, S. cerevisiae, Wheat Germ, or HeLa based cell extracts.

Cell-free biological systems have a wide range of applications in synthetic biology including their use in biosensors, to screen artificial bioparts (such as engineered proteins), and to rapidly prototype genetic circuits (Chappell et al, 2013 and Kelwick et al., 2014). Unlike in vivo systems, cell-free reactions can be directly accessed and therefore have the potential as a platform for the production of proteins and molecules that would otherwise be difficult or impossible to produce in vivo.

We are using a quantitative data-driven approach to rationally optimise cell-free reaction systems.

Sc2.0 - Nuclear Organisation in Yeast

Team: Mike Clarke

Collaborators: Dr. Tom Ellis

The budding yeast S. cerevisiae is a staple of modern genetic research and has been fundamental in furthering our understanding of how genotype becomes phenotype. Current research has revealed the physical organisation of chromosomes within three-dimensional nuclear space to be an important emergent characteristic that impacts gene expression at many levels of regulation.

A variety of nuclear landmarks such as the nucleolus and telomeres are known to tether to specific regions of the nucleus, and to limit the nuclear space which may be explored by any given section of chromosome. Other chromosomal loci, such as those involved in galactose metabolism, are known to be particularly mobile, and this mobility is known to correlate with expression.

Few studies have looked at the persistence of such behaviour following systemic perturbation, and the impacts of such on gene expression. The aim of this research is to examine the effects of perturbation of inter- and intrachromosomal organisation within the nucleus, using fluorescent confocal microscopy, and to correlate findings with the expression levels of specific genes.

Frontier engineering - Fine chemical pathway design

Team: Dr. Simon Moore

Collaborators: Dr. Karen Polizzi

We are interested in the interface of biology and chemistry for the production of high-value fine chemicals using a synthetic biology design approach. This is an holistic approach that addresses how a substrate to product transformation can be simplified and redirected to enhance productivity. Targets of interest include polylactic acid, fragrances and drug precursors, using techniques such as Golden Gate DNA assembly, cell-free transcription-translation, enzymology, structural biology and liquid chromatography mass spectrometry (LC-MS).

Bioplastic production from waste

Team: Dr. Richard Kelwick and Margarita Kopniczky

Collaborators: Dr. Guy-Bart Stan

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. To this end we are exploring forward-design approaches to optimising P(3HB) production in engineered E. coli. Additionally, to further increase the efficiency and commercial viability of P(3HB) production we are also exploring the utilisation of non-recyclable waste as a low-cost carbon source.

For more information see the Imperial College iGEM team page and read our recently published paper in PLoS ONE.

Plasticity

Recycling waste to plastic: A forward-design approach to increase the production of poly-3-hydroxybutyrate in genetically engineered Escherichia coli from mixed waste.

Exploration of context dependency in a standardised B. subtilis cell-free system

Team: Dr. Richard Kelwick, Dr. Alexander J. Webb and Dr. James T. MacDonald

Collaborators: Prof. Colin Harwood (Newcastle University)

This project will explore context dependency of a panel of Bacillus subtilis promoters characterised in vivo (Building upon Nicolas et al. 2012) and in vitro using a standardised B. subtilis cell free system that we are developing. This project is a part of the Flowers consortium.

flowers

The Flowers Consortium: Creating a UK Infrastructure for Synthetic Biology.

Computational protein design

Team: Dr. James T. MacDonald

Collaborators: Dr. James W. Murray

We have developed new computational protein design methods and applied them to the de novo design of loops with experimental validation.

For more information please see Dr. James MacDonald's homepage.

de novo loop design

De novo computationally designed protein loop. Example computationally designed loop where the cyan loop is the X-ray crystal structure and the pink loop is the computational design.

Regulating translation via RNA binding proteins

Team: Margarita Kopniczky

Recently, various new types of regulators acting at the mRNA and translational levels have been constructed, enriching the toolkit for synthetic circuit design in eukaryotic cells. The aim of this project is to systematically characterise one class of such regulators: RNA-motif binding proteins used as translational repressors is mammalian cells. We investigate the switching characteristics and design rules associated with such regulators.