This project aims to develop an innovative approach to tissue engineering by combining synthetic biology with advanced biomaterials and cell-free expression systems. The goal is to address critical limitations in current 3D cell culture technologies and create more biomimetic tissue models.

Three-dimensional (3D) cell culture systems have significantly advanced our understanding of tissue development and disease progression. However, current technologies face two main challenges:

• Difficulty in creating complex, physiologically relevant biochemical gradients

• Limited control over the spatial and temporal distribution of mechanical properties

Project Description: This project will develop a novel platform combining cell-free expression with advanced technologies in 3D bioprinting. The core concept involves encapsulating cell-free expression machinery from bacteria in hydrogel beads (CFE beads) to allow the targeted production of growth factors, peptide hormones and enzymes from within a 3D-printed scaffold. In this way, we can generate 3D-printed topologies with controllable spatially mechanical properties and growth factor gradients that more closely mimic the complexity of natural tissues.

Objectives

Key objectives include:

1. Designing and optimising cell-free expression systems for targeted enzyme and growth factor production.
2. Characterizing the spatiotemporal patterns of gel cross-linking and growth factor distribution.
3. Develop novel bio-inks which include CFE bead additives.

This interdisciplinary project combines synthetic biology, materials science, and 3D printing. The resulting platform has the potential to revolutionise regenerative medicine by providing a highly controllable, scalable, and reproducible method for creating complex tissue-like environments. Such a system could find applications in drug development, disease modelling, and the production of implantable tissue constructs.

Key References

1. Silverman, A. D., Karim, A. S., & Jewett, M. C. (2020). Cell-free gene expression: an expanded repertoire of applications. Nature Reviews Genetics, 21(3), 151-170.
2. Grigoryan, B., et al. (2019). Multivascular networks and functional intravascular topologies within biocompatible hydrogels. Science, 364(6439), 458-464.

This project aims to engineer Clostridium acetobutylicum to enhance biofuel production by expressing coiled-coil (CC) peptides on its surface to capture high-value chemicals. By combining cutting-edge computational design with experimental biology, we seek to develop a novel approach to improve biofuel production's efficiency and economic viability in Clostridia.

Peptides will initially be designed using computational biology. This will use a wealth of different resources, such as Rosetta and molecular dynamics simulations, to create a suite of coiled-coil peptides with high affinity and selectivity for butanol. We will optimise the pocket shape of these peptides using ligand docking techniques to guide the selection of key residues. Both coarse-grained and all-atom molecular dynamics simulations will be employed to assess the stability of the designed peptides and study the diffusion of target molecules.

Following the computational phase, we will transition to experimental work to develop an efficient surface display system for C. acetobutylicum. This will involve evaluating various membranes which can serve as anchors for the surface display of the peptides.

To verify our designs experimentally, we will initially express the top-performing candidates in model organisms like E. coli BL21. We will assess the binding affinity of the high-value chemical to our peptide sequences using biophysical techniques like isothermal calorimetry (ITC). Circular dichroism (CD) spectroscopy will be used to assess the structure and stability of the de novo-designed peptide. Once the tests have been completed using the BL21 test system, CRISPR will be used to incorporate the gene that encodes the novel peptide into the genome of C. acetobutylicum.

This project aims to demonstrate enhanced high-value chemical capture and production in engineered strains of C. acetobutylicum. We will also develop methods for the controlled release of captured chemicals, a crucial step in making this technology viable for industrial applications. This interdisciplinary project, combining synthetic biology, protein engineering, and biophysical techniques, has the potential to advance the field of biofuel production significantly.

By successfully developing surface-expressed CC peptides for chemical capture, we aim to address one of the key challenges in biofuel production: improving yield and efficiency. This research could pave the way for more economically viable and sustainable biofuel production processes, contributing to the broader goal of transitioning to a bio-based economy.

Key references

1. Meng, Y., et al. (2019). A novel method to increase butanol tolerance of Clostridium acetobutylicum by extracellular expression of antioxidant enzymes. Biotechnology for Biofuels, 12(1), 1-10.
2. Thomas, F., Boyle, A. L., Burton, A. J., & Woolfson, D. N. (2013). A set of de novo designed parallel heterodimeric coiled coils with quantified dissociation constants in the micromolar to sub-nanomolar regime. Journal of the American Chemical Society, 135(13), 5161-5166.