Expanding the complexity of cell-free systems for synthetic cells

Abstract

Cell-Free Protein Synthesis (CFPS) harnesses transcription and translation machinery without the extremely complicated cellular metabolism. Because of its versatility for constructing gene-based biological reactions, CFPS has become a great platform not only for fundamental biology, biotechnology, and biomedical research but also for synthetic biology to build synthetic cells. Escherichia coli (E. coli) CFPS uses E. coli lysate as a source of enzymes for protein production. Although E. coli CFPS has advantages in yield, cost, and preparation simplicity, the background reactions derived from the lysate make it challenging to reconstitute various cellular-like functions. In this dissertation, three drawbacks of E. coli CFPS were improved. Firstly, using a recB E. coli strain significantly improved the protein expression efficiency from linear templates, allowing the faster preparation of gene templates using PCR rather than cloning. Secondly, new luciferase variants were introduced. With a substrate regeneration pathway, a longer-time luminescence was achieved. Finally, gene expression control using anti-oligonucleotides was explored, showing targeted gene inhibitions. Although the products of this dissertation did not solve all the limitations associated with E. coli CFPS, those methods moved CFPS a step forward to a fully controllable system for developing further complicated cell-free tools for synthetic biology.