a-practical-guide-to-phage-robotics-assisted-near-continuous

A Practical Guide to Phage- and Robotics-Assisted Near-Continuous Evolution

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and ...

Massachusetts Institute of Technology

Evolution occurs when selective pressures from the environment shape inherited variation over time. Within the laboratory, evolution is commonly used to engineer proteins and RNA, but experimental constraints have limited the ability to reproducibly and reliably explore factors such as population diversity, the timing of environmental changes and chance on outcomes. We developed a robotic system termed phage- and robotics-assisted near-continuous evolution (PRANCE) to comprehensively explore biomolecular evolution by performing phage-assisted continuous evolution in high-throughput. PRANCE implements an automated feedback control system that adjusts the stringency of selection in response to real-time measurements of each molecular activity. In evolving three distinct types of biomolecule, we find that evolution is reproducibly altered by both random chance and the historical pattern of environmental changes. This work improves the reliability of protein engineering and enables the systematic analysis of the historical, environmental and random factors governing biomolecular evolution.

Systematic molecular evolution enables robust biomolecule discovery.

Enhanced ascorbate peroxidase 2 (APEX2) is a protein generated with directed evolution by Lam et al. that has transformed our understanding of subcellular entities and phenomena. The rapid kinetics of this engineered protein highlights the power of directed evolution to expand the molecular toolkit for biologists.

Proximity labeling of endogenous protein interactions enabled by directed evolution.

We report a comprehensive drug synergy study in acute myeloid leukemia (AML). In this work, we investigate a panel of cell lines spanning both MLL-rearranged and non-rearranged subtypes. The work comprises a resource for the community, with many synergistic drug combinations that could not have been predicted a priori, and open source code for automation and analyses. We base our definitions of drug synergy on the Chou-Talalay method, which is useful for visualizations of synergy experiments in isobolograms, and median-effects plots, among other representations. Our key findings include drug synergies affecting the chromatin state, specifically in the context of regulation of the modification state of histone H3 lysine-27. We report open source high throughput methodology such that multidimensional drug screening can be accomplished with equipment that is accessible to most laboratories. This study will enable preclinical investigation of new drug combinations in a lethal blood cancer, with data analysis and automation workflows freely available to the community.

High-throughput approaches to uncover synergistic drug combinations in leukemia.

Liquid handling robots are often limited by proprietary programming interfaces that are only compatible with a single type of robot and operating system, restricting method sharing and slowing development. Here we present PyLabRobot, an open-source, cross-platform Python interface capable of programming diverse liquid-handling robots, including Hamilton STARs, Tecan EVOs, and Opentron OT-2s. PyLabRobot provides a universal set of commands and representations for deck layout and labware, enabling the control of diverse accessory devices. The interface is extensible and can work with any robot that manipulates liquids within a Cartesian coordinate system. We validated the system through unit tests and several application demonstrations, including a browser-based simulator, a position calibration tool, and a path-teaching tool for complex movements. PyLabRobot provides a flexible, open, and collaborative programming environment for laboratory automation.

Article	Year
Systematic molecular evolution enables robust biomolecule discovery. Nature methods\| PubMed ID: 34969982	2022
Proximity labeling of endogenous protein interactions enabled by directed evolution. Trends in biotechnology\| PubMed ID: 36710130	2023
High-throughput approaches to uncover synergistic drug combinations in leukemia. SLAS discovery : advancing life sciences R & D\| PubMed ID: 37121274	2023
PyLabRobot: An Open-Source, Hardware Agnostic Interface for Liquid-Handling Robots and Accessories. bioRxiv : the preprint server for biology\| PubMed ID: 37502883	2023

Stefan Golas

Biography

JoVE Journal Publications

Other Publications