RT Journal Article
SR Electronic
T1 Biofoundry-assisted expression and characterisation of plant proteins
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.03.11.434954
DO 10.1101/2021.03.11.434954
A1 Quentin M. Dudley
A1 Yao-Min Cai
A1 Kalyani Kallam
A1 Hubert Debreyne
A1 Jose A. Carrasco Lopez
A1 Nicola J. Patron
YR 2021
UL http://biorxiv.org/content/early/2021/03/11/2021.03.11.434954.abstract
AB Many goals in synthetic biology, including the elucidation and refactoring of biosynthetic pathways and the engineering of regulatory circuits and networks, require knowledge of protein function. In plants, the prevalence of large gene families means it can be particularly challenging to link specific functions to individual proteins. However, protein characterisation has remained a technical bottleneck, often requiring significant effort to optimise expression and purification protocols. To leverage the ability of biofoundries to accelerate design-built-test-learn cycles, we present a workflow for automated DNA assembly and cell-free expression of plant proteins that accelerates optimisation and enables rapid progression to characterisation. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using E. coli or wheat germ lysates as well as a set of N- and C-terminal tag parts for detection, purification, and improved expression/folding. We next optimised automated assembly of miniaturised cell-free reactions using an acoustic liquid handling platform and then compared tag configurations to identify those that increase expression. We additionally developed a luciferase-based system for rapid quantification that requires a minimal 11 aa tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional characterisation experiments can be performed with cell-free protein synthesis reactions without the need for protein purification. Together, the combination of automated assembly of DNA parts and cell-free expression reactions should significantly increase the throughput of experiments to test and understand plant protein function and enable the direct reuse of DNA parts in downstream plant engineering workflows.Competing Interest StatementThe authors have declared no competing interest.