Circular RNAs to make spider silk

It has long been recognised that spider silk has physical properties that are attractive for industrial applications. For example dragline silk is five times stronger, and more elastic, than steel. Each of the seven types of spider silk have distinct physical properties with potential commercial applications. This proposal aims to simplify silk expression by combining a number of molecular processes to generate a novel and adaptable system for the in vitro and in vivo synthesis of large repetitive proteins in bacterial and eukaryotic systems. The methods developed will also have application in the synthesis of long repetitive proteins for drug delivery or as multimers of therapeutic peptides, cleavable by specific proteases. Circular RNA templates will be designed based on a combination of safe and experimental molecular approaches: ligation of linear RNA molecules, a well characterised and technically straightforward method; self-circularisation using ribozyme activity from self-splicing introns (a technique with huge potential for industrial applications, but which needs developing and optimising).
Long repetitive proteins have huge potential. Proteins such as spider silks have incredible physical properties; peptide drugs could be produced as inactive precursors; molecular sponges or decoys could be synthesised. However, long repetitive genes are difficult to manipulate, and pose significant barriers to efficient protein expression. Based on our expertise and experience in post-transcriptional regulation of gene expression, we have designed protocols for synthesising circular mRNAs. These can involve simply the ligation of a custom designed RNA molecule, which offers a safe mechanism for testing and developing circular mRNA templates. However, more excitingly, we have also designed protocols exploiting the ability of RNA sequences derived from self-splicing introns to catalyse their own circularisation. This offers the opportunity to develop large-scale in vitro expression systems based on mRNAs that can self assemble from simple RNA precursors. The applications for this type of technology are wide ranging: from molecular sensors to therapeutics to novel materials to molecular sponges or decoys. While we have some clear ideas of proteins we'd like to synthesise, we also think that is important for the student to direct the outcomes of this research to align with their own longer term career and research plans. The PhD project will focus initially on using circular RNAs to develop a new type of flexible and adaptable expression system. Building on previous work in the lab, and also on their own work during the rotation project, students will combine standard molecular biology tools with cutting edge RNA biology applications to engineer plasmid constructs for bacterial and eukaryotic expression of proteins. Proof-of-principle and optimisation of circular RNAs will employ in vitro bacterial and eukaryotic expression systems. Later in the project the student will also have the opportunity to develop in vivo expression sytems, in micro-organisms and in cultured eukaryotic cells. Bacteria, yeast and mammalian cells will be transfected with DNA encoding self-circularising RNA and the effects on cells, and the yields and biophysical properties of the proteins produced will be analysed.