Biofoundry-assisted expression and characterization of plant proteins
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 characterization has remained a technical bottleneck, often requiring significant effort to optimize 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 optimization and enables rapid screening of enzyme activity. First, we developed a phytobrick-compatible Golden Gate DNA assembly toolbox containing plasmid acceptors for cell-free expression using Escherichiacoli 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 optimized automated assembly of miniaturized 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–amino acid tag and demonstrate facile removal of tags following synthesis. Finally, we show that several functional assays 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.
{'lacZ-alpha', 'lacZ beta-galactosidase alpha peptide'}
beta-lactamase, ampicillin resistance (ampR)
pUC origin of replication (derivative of pBR322/pMB1/ColE1)
diadenosine tetraphosphatase (apaH) C-terminal fragment ( aa 198-280) AVI54590.1 from E coli MG1655
{"aminoglycoside O-phosphotransferase APH(3')-Ia, kanamycin resistance (kanR)", 'KanR'}
backbone from pJL1-sfGFP (Addgene #69496)
NET tag (N-terminal Expression Tag), MEKKI, from chloramphenicol acetyltransferase, minimizes secondary structure with RBS and 5' UTR
efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)