Solid-binding peptides (SBPs): versatile molecular linkers
Solid-binding peptides (SBPs) are short amino acid sequences that can act as molecular linkers to direct the orientated immobilisation of proteins onto solid materials without impeding their biological activity. Cell-free synthetic biology circumvents many of the limitations encountered by in vivo synthetic biology by operating without the constraints of a cell. It offers higher substrate and enzyme loading and the facile optimisation of enzyme ratios. Some of the challenges of this approach include costly enzyme preparation, biocatalyst stability, and the need for constant supplementation with co-factors. To overcome these challenges, we have developed a molecular toolbox that facilitates the construction of biocatalytic modules with predefined functions and catalytic properties. It consists of three interchangeable building blocks: (a) low-cost inorganic matrices (e.g., silica, zeolite), (b) matrix-specific SBPs and (c) thermostable enzymes. The rational combination of these building blocks allows for flexibility and a ‘pick, mix’ and re-use’ approach with multiple biocatalytic modules available for the assembly of natural and non-natural pathways. Individual immobilised enzymes can be combined rationally to assemble recyclable and product-specific reactions.
Cell-free biosynthesis of platform chemicals from organic waste
In the last decades microbial biotransformation has replaced costly and difficult chemical synthesis of a wide range of chemicals. Metabolic engineering of microbial hosts, however is a time consuming, laborious and highly complex task. Major challenges such as high substrate costs, cell viability and product toxicity limit the possibilities of in vivo metabolic engineering. A cell free approach of the conversion from natural feedstocks via selected biocatalysts has the potential to become a powerful strategy in tackling the increasing demand of bulk and speciality chemicals. By heterologous expression of selected or engineered enzymes, near-endless combinations of highly efficient biosynthetic routes are possible. In our lab we isolate, characterize and immobilise novel enzymes, for the assembly of efficient production pathways for useful chemicals. Years of extensive research on cellulytic and hemicellulytic enzymes aid to break down complex sugars from various waste compounds. These sugar monomers are being converted by nearly theoretical yield into desired valuable compounds.
Protein-based nanoparticles for drug delivery
Compartmentalisation is an important organisational feature of life that allows otherwise incompatible biochemical processes to function cohesively within a cell. It occurs at varying levels of complexity, from eukaryotic organelles and bacterial microcompartments, to viral capsids and even the molecular reaction chambers formed by enzyme assemblies. Encapsulins are a newly reported class of protein-based nanocompartments produced in bacteria and archaea. They are typically composed of multiple copies of a single protein subunit, which self-assemble with precision to form hollow cage-like nanostructures that are uniform in composition, size and morphology. Encapsulins have been recently used to encapsulate foreign cargo, such as recombinant proteins and inorganics. In addition, the external and internal surfaces of these nanocompartments can be easily genetically engineered to display short peptide sequences (e.g. epitopes for vaccines, peptide-based drugs, and antimicrobial peptides) that can further enhance their functionality. Accordingly, encapsulins represent a promising alternative to the lipid, polymer, and inorganic-based compartments that are currently used as vehicles for the encapsulation and targeted delivery of therapeutics. This project will use synthetic biological techniques to modify encapsulins that can be loaded with a drug and then upon reaching their biological target be activated to disassemble and release the drug, thus providing both spatial and temporal control of drug delivery in vivo. This project is lead by Dr Andrew Care.
Hand-held convective PCR
Molecular diagnostic methods like real-time polymerase chain reaction (PCR) and multiplex PCR are considered reliable for identification and detection of nucleic acids from biological samples. However, they are poorly suited for implementations in remote clinical settings because of their requirement for dedicated laboratory space, cost, electricity and highly-trained personnel. One of the promising solutions for rapid and simple DNA amplification is the use of Rayleigh–Bernard natural convection, which is caused by buoyancy driven thermal gradient of liquid when heated from below. The natural convection avoids the use of complex and sophisticated hardware that is required for precise maintenance of temperature cycles in conventional PCR. This drastically reduces the cost and time require for amplification of target DNA. In this project, a hand-held convective PCR device was developed for rapid isothermal detection of nucleic acid. The specific detection of amplicons was achieved using magnetic bead-based hybridization assay. This study integrates the simplicity of convective PCR and the specificity of magnetic bead-based assay for rapid detection of nucleic acid. The magnetically-captured DNA are hybridized to fluorescently-labelled DNA probe for specific detection of the target DNA which can be extended for multiplexing by introducing different fluorophore-labelled DNA probes. Thus, the integration of convective PCR with magnetic bead-based hybridization assay could enable high throughput molecular testing of nucleic acids in resource limited settings. The simplicity of convective PCR provides immense potential for rapid point-of-care molecular diagnostic applications.