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 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.
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's tail coat protein, which is needed for phage propagation, this 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 "near-continuous" 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.
Despite efforts to standardize equipment, practically speaking, every PRANCE setup will be different due to changes in equipment supply, hardware, and software versioning. As a result, each PRANCE setup manifests unique setup challenges, demanding a comprehensive understanding of the purpose of each component for effective modular troubleshooting.
This method delineates a step-by-step protocol for the setup and testing of an established PRANCE system. We first focus on the critical elements of the hardware and software and then detail the essential steps to prepare for and conduct a series of test runs, which establish that the system is ready for PRANCE.
An essential feature of the hardware is optimization to reduce the risk of sample cross-contamination during multiplexed experiments using bacteriophage. It is recommended to use exclusively filtered tips with robot tip technology that is compatible with tip reuse and is thought to minimize aerosols produced during tip ejection by avoiding force-fit tips. Robust tip washing as per this protocol allows for tip reuse although the adequacy of this must be validated as part of the infection test on each system. Self-sterilization is also dependent on a consistent supply of water and bleach for the system. These are stored in tanks/buckets and if depleted will result in impaired self-sterilization and rapid cross-contamination. Photographs can be taken of the tanks/buckets taken before and after the program runs to benchmark the rate at which the washing equipment consumes water and bleach given a particular pump setup.
Another key element of the system is the maintenance of the bacterial growth phase and temperature. PRANCE experiments are conducted using the S2060 E. coli bacterial strain (Addgene: #105064). This is a K12-derived F-plasmid-containing strain optimized to reduce biofilms7. In addition, the F-plasmid in this strain has been edited with the addition of a tetracycline resistance cassette for plasmid maintenance, luxCDE and luxR to complement luxAB-mediated luminescence monitoring, as well as lacZ under the phage shock promoter to allow for colorimetric visualization of plaques. The F-plasmid-encoded F-pilus is necessary for M13 phage infection. Bacteria used in PACE must therefore be cultured at 37 °C and at mid-log phase when the F-pilus12 is expressed and M13 phage infection, propagation, and evolution are possible. For static temperature regulation, an off-the-shelf heated plate carrier can be employed. An alternative is simply heating the air going into the HEPA filter using inexpensive heaters, though this is not recommended as it may lead to accelerated wear and tear on the hardware. In addition, this accelerates the evaporation of auxiliary on-deck fluids, such as the bleach/water buckets and inducer, when used.
Calibration of the software packages is also essential for proper system function. Divergences between the software deck layout and the actual robot deck are the most common cause of system failure during operation. Regular calibration of the auxiliary pumps that supply bacterial culture, bleach, and drain the system is vital as peristaltic pump usage can lead to tubing wear and fluid volume alterations.
The water run test will rapidly reveal a number of common setup problems, including incorrect liquid handling settings, fluidics leaks/faulty connections, and software instability. A successful water run will exhibit no unexpected liquid leaks and run stably without errors overnight. There are a number of common issues that may arise during a water run such as failure to execute certain liquid-handling steps, dripping from pipettes, and the protocol stopping mid-run. In case of failure to execute certain liquid handling steps, confirm that all liquid classes have been installed. These list the appropriate viscosity and pipetting speeds and are adjusted in the robot control software provided by the manufacturer. If there is dripping from pipettes, it is important for robot pipetting arm settings to be correct to enable clean pipetting and eliminate phage cross-contamination. Successful robotic pipetting requires, in addition to correct liquid classes, correct deck layout heights of all labware, and appropriate pipetting height offsets specified in the PRANCE robot method program. These height offsets may require direct adjustment. If the protocol stops mid-run, often this will be generated by a wide array of errors that indicate that the deck layout file may not match the actual deck configuration.
The bacteria-only run test will reveal issues with plate reader settings and real-time data visualization, problems with excessive bleach concentration or insufficient rinsing, and temperature stability. A successful bacteria-only run will exhibit equilibration of lagoon absorbance over the first three cycles, followed by stable absorbance for the duration of the run. In addition, it may reveal several common issues. This is the first step where the data generated by the plate reader are plotted. Data in the plate reader database may not be saved properly or plotted properly. If bacteria fail to equilibrate in their absorbance, this may indicate that the bleach concentration is too high. Excessive bleach or insufficient washing can sterilize the entire experiment, rather than just the piece of labware. If this is suspected, bleach-detecting strips can be used to test the lagoon. The stability of the temperature of the culture can be checked with a thermometer gun.
A successful infection test indicates that the system is ready for PRANCE runs. An infection test can be performed by inoculating a subset of lagoons containing bacterial culture. These bacteria will express pIII when infected by the appropriate phage that lacks the gene for pIII (ΔgIII), allowing phage propagation. One possible combination for testing is to use S2060 bacteria transformed with a plasmid expressing pIII under the phage shock promoter with any ΔgIII phage. We recommend using ΔgIII phage bearing the wild-type T7 RNA Polymerase with S2060 bacteria transformed with an accessory plasmid, in which pIII and luxAB are driven by the T7 promoter (Plasmid pJC173b13), as illustrated in Figure 1. This also allows plate-reader-mediated monitoring of infection during the test run. Definitive evidence of the success of the infection test and lack of cross-contamination will come from phage titering of test and control lagoons. Where a luciferase reporter is used, an increase in luminescence in test wells only, as seen in Figure 3, is also an indicator of successful phage infection and propagation. The gold standard for phage titer quantification is the plaque assay7. There is also a protocol for M13 quantification by qPCR7 that may be faster, although this does not discriminate between infectious and non-infectious phage particles and thus may overestimate titers.
The main program references a manifest file, this is a plain text database file, which dictates the dilution volume per cycle of each propagating culture as well as the selection of any number of potential bacterial culture feedstocks, which may differ in selection stringency. In this manner, the manifest file defines many of the parameters of the PRANCE run. It should be noted that this file can be edited during the run by either the operator or the system, meaning that manual or automatic feedback control can be effected.
The utility of a fully functioning PRANCE setup lies in its capacity to rapidly evolve large populations in a carefully monitored and controlled environment. The plate-based format distinguishes PRANCE from other techniques, like using smaller off-the-shelf turbidostat-based systems14,15. The plate-based setup not only facilitates easy integration with additional robotic processing steps but also compatibility with other laboratory instruments such as centrifuges. Moreover, the ability to conduct accelerated evolution concurrently across multiple instances introduces an additional dimension to the experiment, enhancing the prospect of achieving diverse and robust results. The granular control and feedback system integral to PRANCE further bolsters the predictability and reliability of the experiment, marking a significant advancement in the field of directed evolution techniques. However, this technique is limited in the number of parallel experiments it can conduct. Depending on the configuration, PRANCE setups are usually limited either by robot pipetting speed or by available deck space.
The same hardware and software used for PRANCE can also be applied to evolution methods that do not involve bacteriophage. As demonstrated in the many-turbidostats method11, this same instrument can be employed exclusively with bacteria, enabling whole-genome adaptive evolution experiments. This adaptability widens the scope of this instrument, paving the way for new forms of Robotics-accelerated Evolution.
The authors have nothing to disclose.
We thank Emma Chory and Kevin Esvelt for their help and advice with hardware and software setup. Samir Aoudjane, Osaid Ather, and Erika DeBenedictis are supported by the Steel Perlot Early Investigator Grant. This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (CC2239), the UK Medical Research Council (CC2239), and the Wellcome Trust (CC2239).
3D printed bacterial reservoir "waffle" | – | – | https://drive.google.com/file/d/16ELcvfFPzBzNSto0xUrBe-shi23J9Na7/view; For Robot deck |
3D printer | FormLabs | Form 3B+ | 3D printer components |
3D printer resin (clear) | FormLabs | RS-F2-GPCL-04 | consumable for 3D printer |
8-1,000 µL head | Hamilton | 10140943 | For Liquid handling robot |
96-1,000 µL pipetting head | Hamilton | 10120001 | For Liquid handling robot |
Black polystyrene plate reader microplates | Millipore Sigma | CLS3603 | For Robot deck |
BMG Labtech Spectrostar FLuorstar Omega | BMG Labtech | 10086700 | For Liquid handling robot |
Cleaning solution | Fluorochem Limited | F545154-1L | used to clean the liquid handling parts of the robot |
Deep Well plates | Appleton Woods | ACP006 | these are used to contain evolving bacteria on the deck of the robot |
encolsure heater | Stego | 13060.0-01 | heats inside robot enclosure |
Hamilton STAR | Hamilton | 870101 | For Liquid handling robot |
Heater | Erbauer | BGP2108-25 | For Liquid handling robot |
HIG Bionex centrifuge | Hamilton | 10086700 | For Liquid handling robot |
iSWAP plate gripper | Hamilton | 190220 | For Liquid handling robot |
laboratory tubing | Merck | Z280356 | to construct liquid handling manifold |
luer to barb connector | AIEX | B13193/B13246 | for connectorizing tubing |
Magnetic stir plate | Camlab | SKU – 1189930 | For Auxiliary Fridge |
Molcular pipetting arm | Hamilton | 173051 | For Liquid handling robot |
Omega | BMG labtech | 5.7 | plate reader control software |
One way Check Valves | Masterflex | MFLX30505-91 | to one way sections of liquid handling manifold |
pyhamilton | MIT/Open source | https://github.com/dgretton/std-96-pace%20PRANCE | open source python robot control software |
pymodbus | opensource | 3.5.2 | python pump software interface |
Refrigetator | Tefcold | FSC175H | allows cooled bacteria to be used instead of turbidostat |
S2060 Bacterial strain | Addgene | Addgene: #105064 | E. coli |
temperature controller | Digiten | DTC102UK | Used to control heaters thermostatically |
Thermostat switch controller | WILLHI | WH1436A | WILLHI WH1436A 10 A Temperature Controller 110 V Digital Thermostat Switch Sous Vide Controller NTC 10K Sensor Improved Version; for Liquid handling robot |
Venus | Hamilton | 4.6 | proprietary robot control software |
Wash Station for MPH 96/384 | Hamilton | 190248 | For Liquid handling robot |
Suggested pump manufacturers | |||
Company | Catalog number | Notes | Documentation |
Agrowtek | AD6i Hexa Pump | https://www.agrowtek.com/doc/im/IM_ADi.pdf | |
Amazon | INTLLAB 12V DC | ||
Cole-Parmer | EW-07522-3 | Masterflex L/S Digital Drive, 100 RPM, 115/230 VAC | https://pim-resources.coleparmer.com/instruction-manual/a-1299-1127b-en.pdf |
Cole-Parmer | EW-07554-80 | Masterflex L/S Economy variable-speed drive, 7 to 200 rpm, 115 VAC | https://pim-resources.coleparmer.com/instruction-manual/a-1299-1127b-en.pdf |