pre-run preparation for phage- and robotics-assisted near-continuous evolution &#40;prance&#41; technique

Setting Up PRANCE: A Comprehensive Guide for Robotics-Accelerated Evolution

PRANCE is a combination of two powerful directed evolution techniques. First is PACE1, a molecular technique that couples rounds of gene diversification and selection to the fast life cycle of the M13 bacteriophage, enabling rapid rounds of evolution to occur continuously in liquid phage culture. This selection is driven by the use of a plasmid-encoded gene circuit that couples the function of the evolving protein to the expression of pIII, M13&#39;s tail coat protein, which is needed for phage propagation, this&#160;is illustrated in Figure 1. At the experimental level, continuous dilution of the liquid phage culture allows for continuous selection. Selection stringency can thus be modulated both at the level of the gene circuit as well as at the experimental level by controlling the phage culture dilution rate. PACE can therefore be applied to any biomolecule engineering challenge for which there is a molecular sensor that can detect the desired activity in E. coli bacteria to induce pIII expression. Applications include the evolution of protein-protein binding2,3,4, protein-DNA binding5, protein solubility6, and numerous specific enzymatic functions7. Second is Robotics-accelerated Evolution8,9, which uses a feedback controller to eliminate two common failure modes of directed evolution: extinction, which occurs when the environment is too stringent, and lack of evolution, which occurs when the environment is too lenient. Unlike serial passaging of phage as done in PANCE (Phage-assisted Non-continuous Evolution)7,10, Robotics-accelerated &#34;near-continuous&#34; evolution involves rapid pipetting that maintains cultures at mid-log phase, allowing populations to experience continuous cycles of infection and propagation. When these two technologies are used together, they are referred to as PRANCE, for Phage and Robotics-assisted Near-continuous Evolution8, which enables robust, multiplexed, and rapid continuous evolution. PRANCE has been used to evolve polymerases, tRNAs, and amino-acyl tRNA synthetases and to do feedback control during those evolutions to improve their speed and reliability8.
There are several details of the hardware and software setup for PRANCE that enable the use of bacteriophage on a liquid-handling robot. Instead of using default software provided by the robot manufacturer, we use a python-based open-source software package11, which enables fast, concurrent execution and thus, the ability to keep the semi-continuous bioreactors at mid-log phase. Researcher hands-off time can be extended to several days by having several on-deck components routinely self-sterilize, and this is achieved with automatic control of pumps that can bleach and rinse these components. Phage cross-contamination can be eliminated by the use of a liquid handling robot that does not use force-fit tips and careful adjustment of liquid handling settings.

Author Spotlight: Advancing Protein Engineering &amp;#8211; Harnessing Evolution Through PRANCE and Lab Automation

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

We're trying to make the mysterious process of evolution predictable enough to use for engineering. PRANCE gives us the ability to bring evolution into the lab timescale with parallelization to understand it better. Lab automation is bringing in concepts from more general software engineering, including AI into developing control code.People are also integrating manipulator arms and multi-directional conveyor systems into liquid handling platforms. Overall, automation is including more advanced instrumentation and workflows. Feedback control improves continuous evolution success by preventing common failure modes like extinction.This is essential during multiplex evolutions where molecules have varying starting activities and dynamic evolution stringency is critical for desired results. New scientific questions include exploring how different conditions impact evolution, and looking at the effects on phage propagation and evolution circuit efficiency. With these improved protein engineering tools, we are looking to engineer more sophisticated proteins that are sensitive to their environment and dynamic in function.

Pre-Run Preparation for Phage- and Robotics-Assisted Near-Continuous Evolution &#40;PRANCE&#41; Technique

pre-run-preparation-for-phage-robotics-assisted-near-continuous

Research

JoVE Journal

Bioengineering

190 Aufrufe.  The Francis Crick Institute. Zu Beginn werden Bakterienkulturen in der logaritmischen Phase für das Experiment erworben. Aktualisieren Sie die Controller-Manifestdatei mit Bakterienkulturvolumina für jede 96-Well-Lagune pro Programmzyklus. Verwenden Sie den Verdünnungsrechner XLSX, um die Verdünnungsrate der Lagune zu berechnen.Stellen Sie die variable feste Lagunenhöhe in der Robotermethode PY auf den Standardwert von 14 Millimetern ein, was einem Lagunenvolumen von 550 Mikrolitern entspricht. Platzieren Sie...

Serie: Vorbereitung vor dem Lauf für die Phagen- und Robotik-unterstützte Near-Continuous-Evolution-Technik (PRANCE)

Robotics-accelerated Evolution techniques improve the reliability and speed of evolution using feedback control, improving the outcomes of protein and organism evolution experiments. In this article, we present a guide to setting up the hardware and software necessary to implement Phage- and Robotics-assisted Near-continuous Evolution (PRANCE). PRANCE combines fast phage-based molecular evolution with the ability to run hundreds of independent, feedback-controlled evolution experiments simultaneously. This paper will describe the hardware requirements and setup for PRANCE, including a liquid-handling instrument, a plate reader, auxiliary pumps, heaters, and 3D-printed containers. We describe how to configure the liquid handling robot to be compatible with Python-based open-source software. Finally, we provide suggestions for the first two experiments that can be conducted with a newly constructed PRANCE system that exercises its capabilities and validates that the system is ready to conduct multiplexed evolution. This guide is intended to serve as a handbook for navigating the considerable equipment setup associated with conducting robotics-accelerated evolution.

Phage- and Robotics-assisted Near-continuous Evolution (PRANCE) is a technique for rapid, robust protein evolution. Robotics allows the parallelization of experiments, real-time monitoring, and feedback control.

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