Biofoundry-assisted expression and characterization of plant proteins

Area of Science:

• Synthetic Biology
• Plant Biotechnology
• Protein Engineering

Background:

• Understanding plant protein function is crucial for synthetic biology applications like pathway engineering.
• Large gene families in plants complicate the assignment of specific functions to individual proteins.
• Protein characterization is a bottleneck due to challenges in optimizing expression and purification.

Purpose of the Study:

• To develop an automated workflow for DNA assembly and cell-free expression of plant proteins.
• To accelerate the optimization of protein expression and enable rapid screening of enzyme activity.
• To facilitate the design-build-test-learn cycles in synthetic biology.

Main Methods:

• Developed a phytobrick-compatible Golden Gate DNA assembly toolbox for cell-free expression (E. coli and wheat germ lysates).
• Optimized automated assembly of miniaturized cell-free reactions using acoustic liquid handling.
• Compared N- and C-terminal tag configurations for improved expression and developed a luciferase-based quantification system.

Main Results:

• Successfully automated DNA assembly and cell-free protein expression for plant proteins.
• Identified optimal tag configurations for enhanced protein expression and facilitated tag removal.
• Demonstrated functional assays with cell-free protein synthesis reactions without prior purification.

Conclusions:

• The automated workflow significantly increases experimental throughput for plant protein characterization.
• This approach enables rapid screening of enzyme activity and understanding of protein function.
• Facilitates the direct reuse of DNA parts in downstream plant engineering workflows.