21ENGBIO Engineered orthogonal ribosomes for programmable protein modification

Proteins are tiny nano-scale molecular machines that act as the workhorses of all living cells. They underpin crucial tasks spanning sensing and signalling, the coordination of metabolism and even the self-assembly of structural elements of the cell. Many of these functions can be tailored by the modification of the proteins involved, offering a way for a cell to diversity its behaviour. The broad applications of proteins in biological systems makes them an important target for engineering new forms of biology or harnessing biological components and functions in other areas like Material Science. Being able to synthesise and modify proteins on demand could unlock this huge potential.

In this project we aim to directly tackle this challenge by creating what is termed an "orthogonal ribosome" that can synthesise proteins in parallel to a cell's native process. Importantly, our orthogonal ribosomes will be engineered to include attachment points for secondary components that are able to modify the protein being synthesised. By synthesising our proteins with orthogonal machinery, we avoid modifying native cellular proteins in a detrimental way and thus have the freedom to modify our own in diverse ways. Furthermore, by switching the modifying attachment that is present, we can easily change the type of modification made, creating a platform for programmable protein synthesis and modification.

To achieve this ambitious goal, we will use newly developed experimental methods that can create vast numbers of orthogonal ribosome designs with different attachment points and assess the impact these have on the ability for the ribosome to effectively synthesise a protein. Those designs that work well will be selected and then modifying attachments precisely designed using computer models and simulation to have shapes that ensure the region involved in modification is perfectly positioned on the ribosome. Finally, we will combine the engineered orthogonal ribosomes and modifying attachments within living cells and test their ability to modifying a target protein such that it becomes localised to the edge of a cell when altered - a change we will be able to easily monitor using single-cell microscopy.

This project is an attempt to develop the new methods needed to engineer the complex biological process of protein synthesis through the "augmentation" of a native biomolecular machine - the ribosome. Our flexible and modular approach using "plug-n-play" components offers the ability to rapidly alter the modifications made to a target protein without the need to build a new system from scratch, and opens opportunities for Biologists, Biological Engineers, and Material Scientists to better understand the function of proteins in their native context, precisely engineer their properties in living cells, and make use of highly modified proteins as nanoscale building blocks for new forms of sustainable, high-performance material. More broadly, our methodology also offers a path to harnessing other core cellular processes and repurposing their functionalities for novel applications in the emerging area of Engineering Biology.

Proteins act as both molecular machines and physical building blocks of living cells and carry out a wide variety of functions. Modification of proteins further diversifies their functionalities. Thus, the ability to synthesise a target protein with programmable modifications would open avenues to better understand fundamental biological processes, as well as act as a substrate with which to build future biotechnologies and protein-based biomaterials in Engineering Biology.

As a step towards this goal, this project will develop a flexible platform for the production and targeted modification of proteins in living cells. To avoid potentially detrimental interactions with native translational processes, we will use an orthogonal tethered ribosome (oRibo-T) that is able to translate messenger RNAs that are not recognised by the host cell. Our oRibo-T will be engineered have additional protein attachment sites introduced by transposase mutagenesis, and high-throughput cell sorting and sequencing used to screen for oRibo-T variants where the attachment site has minimal impact on protein translation efficiency. Then, using computational protein design, we will develop protein-based modifying attachments consisting of an attachment domain, linker and N-myristoyltransferase domain positioned at the exist tunnel of the ribosome to allow for myristoylation of the ribosome-nascent protein chain. Precise positioning will be achieved by using a modular protein linker whose geometry is rationally designed using computational modelling. Finally, we will bring together our engineered oRibo-Ts with their complementary modifying attachments in Escherichia coli cells, using a fluorescent reporter protein as a target whose localisation to the cell membrane is induced by myristoylation. Efficiency of protein modification will be assessed in vivo via single-cell fluorescence microscopy and further verified using mass spectrometry and fluorescence polarisation assays.