APEX: Automated Protein EXpression in Escherichia coli.

Area of Science:

• Biotechnology
• Molecular Biology
• Microbial Engineering

Background:

• Escherichia coli (E. coli) is a primary host for recombinant protein production due to its rapid growth and genetic tractability.
• Existing E. coli protein expression protocols are often time-consuming, resource-intensive, and prone to operator error, hindering high-throughput applications.
• There is a need for automated, reproducible, and user-friendly systems for efficient heterologous protein expression in E. coli.

Purpose of the Study:

• To develop and validate an automated pipeline, Automated Protein EXpression (APEX), for high-throughput recombinant protein production in E. coli.
• To leverage the Opentrons OT-2 platform for precise and reproducible automation of microbial handling and protein expression workflows.
• To create a customizable and user-friendly system adaptable to diverse research needs without requiring coding expertise.

Main Methods:

• Development of the APEX pipeline utilizing the Opentrons OT-2 liquid handling platform.
• Configuration of APEX for automated heat shock transformation, selective plating, colony sampling, and microculturing.
• Automation of protein expression induction and downstream processing steps within the APEX framework.
• Validation of automated transformation with plasmids ranging from 2.7-17.7 kb and expression of proteins from 27-222 kDa.

Main Results:

• Successful implementation of APEX for automated recombinant protein production in E. coli with high precision and reproducibility.
• Demonstration of APEX's capability to handle diverse plasmid sizes and express a wide range of protein molecular weights.
• APEX effectively automates key steps including transformation, plating, colony selection, and induction, reducing manual labor and potential errors.
• The system requires minimal hardware modifications to the OT-2 platform, making it cost-effective and accessible.

Conclusions:

• APEX provides a robust, automated solution for high-throughput recombinant protein expression in E. coli.
• The automated pipeline significantly improves efficiency, reproducibility, and accessibility compared to traditional methods.
• APEX is a modular and user-friendly platform that can be readily adopted by researchers without specialized programming skills.
• The open-source availability of APEX facilitates its widespread use and further development in the scientific community.