{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T15:19:42Z","timestamp":1785424782818,"version":"3.56.0"},"posted":{"date-parts":[[2026,3,3]]},"group-title":"Bioengineering","reference-count":60,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,3,3]],"date-time":"2026-03-03T00:00:00Z","timestamp":1772496000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/02pjt4q95","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F32-GM157893"],"award-info":[{"award-number":["F32-GM157893"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32-GM144291"],"award-info":[{"award-number":["T32-GM144291"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,3,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Natural evolution is high-dimensional; organisms adapt to many pressures at once, across substrates, environments, and genetic backgrounds. Yet most directed evolution methods flatten this landscape to a single selection axis, hiding tradeoffs, and limiting what can be learned. Phage-assisted continuous evolution (PACE) is uniquely suited for multivariate selection because horizontal gene transfer couples genotype to propagation and allows the same phage lineage to traverse different selection environments. In practice, implementing this at scale has been prohibitive because each selection demands its own host culture, and every culture must be held for days to weeks within a narrow, infectable density window using continuously responsive bioreactors. In this work, TurboPRANCE is presented as an open-source, queueable robotic platform that integrates \u223c200 independently controlled turbidostats with 96 parallel PACE lagoons under closed-loop control. Each turbidostat operates as a fully separate unit that can be equilibrated and initiated on its own schedule, enabling asynchronous starts and sustained operation without intervention. Automated media formulation, programmable dosing, on-deck sterilization, and adaptive scheduling coordinate growth control with the changing needs of the robotic workflow, dynamically adjusting dilution and transfer timing around formulation, sampling, and handling steps to keep each culture at consistent infectable densities despite unpredictable method demands. Cultures can be multiplexed and titrated into lagoons at defined ratios, swapped in and out on a schedule, or kept fully separate across experiments, creating a combinatorial space of selection pressures and programs that is effectively unbounded. Additionally, to enable high-throughput evolutionary tracking that scales with TurboPRANCE, Nanopore long-read sequencing was combined with DeepVariant, a deep learning-based variant caller, enabling population-level tracking of evolving variants. The result is a system that generates high-resolution time-resolvable evolutionary trajectories and large parallel datasets spanning diverse selection regimes, yielding dense, multivariate training data to map and engineer complex fitness landscapes at scale.<\/jats:p>\n                <jats:sec>\n                  <jats:title>Abstract Figure<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" fig-type=\"figure\" orientation=\"portrait\">\n                    <jats:caption>\n                      <jats:title>Turbidostat, Phage, and Robotics-Assisted Near Continuous Evolution (TurboPRANCE)<\/jats:title>\n                      <jats:p>In phage-assisted continuous evolution (PACE), biomolecular activity is coupled to pIII expression, linking function to phage propagation. By altering the host strain, distinct selection pressures can be imposed on the same evolving phage population. In TurboPRANCE, \u223c200 selection programs can vary over time, including periodic \u201cdrift\u201d (mutagenesis), alternation between pressures, or rotational reassignment of host sources, enabling a combinatorial space of selection pressures.<\/jats:p>\n                    <\/jats:caption>\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"709196v2_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.03.02.709196","type":"posted-content","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T01:45:16Z","timestamp":1772588716000},"source":"Crossref","is-referenced-by-count":0,"title":["An autonomous system for multi-objective continuous evolution at scale"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0670-3236","authenticated-orcid":false,"given":"Ryan M","family":"Boileau","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"},{"name":"Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA"},{"name":"Center for Quantitative Biodesign, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9620-4347","authenticated-orcid":false,"given":"Stefan M","family":"Golas","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"},{"name":"Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianni","family":"Ma","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bowen","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-0890-0583","authenticated-orcid":false,"family":"Aradhana","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7623-2567","authenticated-orcid":false,"given":"Mary","family":"Jia","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nika","family":"Ilieva","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aidan","family":"Baydush","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-9290-8932","authenticated-orcid":false,"given":"Hanyu","family":"Fu","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8541-9289","authenticated-orcid":false,"given":"Emma J","family":"Chory","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC, USA"},{"name":"Center for Advanced Genomic Technologies, Duke University, Durham, NC, USA"},{"name":"Center for Biomolecular and Tissue Engineering, Duke University, Durham, NC, USA"},{"name":"Center for Quantitative Biodesign, Duke University, Durham, NC, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026031009151574000_2026.03.02.709196v2.1","doi-asserted-by":"publisher","DOI":"10.1038\/nrg3927"},{"key":"2026031009151574000_2026.03.02.709196v2.2","doi-asserted-by":"publisher","DOI":"10.1038\/nature09929"},{"key":"2026031009151574000_2026.03.02.709196v2.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41596-020-00410-3"},{"key":"2026031009151574000_2026.03.02.709196v2.4","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9425"},{"key":"2026031009151574000_2026.03.02.709196v2.5","doi-asserted-by":"crossref","first-page":"102","DOI":"10.1038\/s41592-024-02504-2","article-title":"EvoAI enables extreme compression and reconstruction of the protein sequence space","volume":"22","year":"2025","journal-title":"Nat. Methods"},{"key":"2026031009151574000_2026.03.02.709196v2.6","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms6352"},{"key":"2026031009151574000_2026.03.02.709196v2.7","doi-asserted-by":"publisher","DOI":"10.1126\/science.abf5972"},{"key":"2026031009151574000_2026.03.02.709196v2.8","doi-asserted-by":"crossref","first-page":"1547","DOI":"10.1002\/cbic.201800707","article-title":"Evolution of C-terminal modification tolerance in full-length and split T7 RNA polymerase biosensors","volume":"20","year":"2019","journal-title":"Chembiochem"},{"key":"2026031009151574000_2026.03.02.709196v2.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-26279-8"},{"key":"2026031009151574000_2026.03.02.709196v2.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-018-0121-5"},{"key":"2026031009151574000_2026.03.02.709196v2.11","doi-asserted-by":"crossref","first-page":"1720","DOI":"10.1038\/s41592-025-02740-0","article-title":"PANCS-Binders: a rapid, high-throughput binder discovery platform","volume":"22","year":"2025","journal-title":"Nat. Methods"},{"key":"2026031009151574000_2026.03.02.709196v2.12","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3515"},{"key":"2026031009151574000_2026.03.02.709196v2.13","doi-asserted-by":"crossref","first-page":"26957","DOI":"10.1021\/acsomega.0c03508","article-title":"Phage-assisted continuous evolution (PACE): A guide focused on evolving protein-DNA interactions","volume":"5","year":"2020","journal-title":"ACS Omega"},{"key":"2026031009151574000_2026.03.02.709196v2.14","doi-asserted-by":"publisher","DOI":"10.1021\/acssynbio.8b00481"},{"key":"2026031009151574000_2026.03.02.709196v2.15","doi-asserted-by":"crossref","first-page":"1581","DOI":"10.1021\/acscentsci.1c00811","article-title":"Phage-assisted continuous evolution and selection of enzymes for chemical synthesis","volume":"7","year":"2021","journal-title":"ACS Cent. Sci"},{"key":"2026031009151574000_2026.03.02.709196v2.16","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.2474"},{"key":"2026031009151574000_2026.03.02.709196v2.17","doi-asserted-by":"crossref","first-page":"588","DOI":"10.1038\/s41589-024-01823-x","article-title":"Rapid discovery of cyclic peptide protein aggregation inhibitors by continuous selection","volume":"21","year":"2025","journal-title":"Nat. Chem. Biol"},{"key":"2026031009151574000_2026.03.02.709196v2.18","doi-asserted-by":"crossref","first-page":"4202","DOI":"10.1038\/s41467-020-18018-2","article-title":"Continuous bioactivity-dependent evolution of an antibiotic biosynthetic pathway","volume":"11","year":"2020","journal-title":"Nat. Commun"},{"key":"2026031009151574000_2026.03.02.709196v2.19","doi-asserted-by":"publisher","DOI":"10.1038\/nature17938"},{"key":"2026031009151574000_2026.03.02.709196v2.20","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.07.039"},{"key":"2026031009151574000_2026.03.02.709196v2.21","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0193-0"},{"key":"2026031009151574000_2026.03.02.709196v2.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0453-z"},{"key":"2026031009151574000_2026.03.02.709196v2.23","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-45969-7"},{"key":"2026031009151574000_2026.03.02.709196v2.24","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01410-2"},{"key":"2026031009151574000_2026.03.02.709196v2.25","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-023-01427-x"},{"key":"2026031009151574000_2026.03.02.709196v2.26","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkae534"},{"key":"2026031009151574000_2026.03.02.709196v2.27","first-page":"22","article-title":"Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing","volume":"9","year":"2025","journal-title":"Nat. Biomed. Eng"},{"key":"2026031009151574000_2026.03.02.709196v2.28","doi-asserted-by":"publisher","DOI":"10.1126\/science.adt5199"},{"key":"2026031009151574000_2026.03.02.709196v2.29","doi-asserted-by":"publisher","DOI":"10.1038\/s41592-021-01348-4"},{"key":"2026031009151574000_2026.03.02.709196v2.30","doi-asserted-by":"crossref","unstructured":"Xie, V. C. , Pu, J. , Metzger, B. P. , Thornton, J. W. & Dickinson, B. C. Contingency and chance erase necessity in the experimental evolution of ancestral proteins. Elife 10, (2021).","DOI":"10.7554\/eLife.67336"},{"key":"2026031009151574000_2026.03.02.709196v2.31","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1220670110"},{"key":"2026031009151574000_2026.03.02.709196v2.32","doi-asserted-by":"publisher","DOI":"10.1021\/bi3016185"},{"key":"2026031009151574000_2026.03.02.709196v2.33","doi-asserted-by":"publisher","DOI":"10.1126\/science.4001944"},{"key":"2026031009151574000_2026.03.02.709196v2.34","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2006.94"},{"key":"2026031009151574000_2026.03.02.709196v2.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.jim.2007.11.008"},{"key":"2026031009151574000_2026.03.02.709196v2.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2018.10.021"},{"key":"2026031009151574000_2026.03.02.709196v2.37","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1453"},{"key":"2026031009151574000_2026.03.02.709196v2.38","doi-asserted-by":"publisher","DOI":"10.1101\/2025.11.19.689279"},{"key":"2026031009151574000_2026.03.02.709196v2.39","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.4151"},{"key":"2026031009151574000_2026.03.02.709196v2.40","doi-asserted-by":"crossref","first-page":"e9942","DOI":"10.15252\/msb.20209942","article-title":"Enabling high-throughput biology with flexible open-source automation","volume":"17","year":"2021","journal-title":"Mol. Syst. Biol"},{"key":"2026031009151574000_2026.03.02.709196v2.41","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01368-07"},{"key":"2026031009151574000_2026.03.02.709196v2.42","doi-asserted-by":"publisher","DOI":"10.1038\/s41589-021-00876-6"},{"key":"2026031009151574000_2026.03.02.709196v2.43","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-024-10440-w"},{"key":"2026031009151574000_2026.03.02.709196v2.44","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.4235"},{"key":"2026031009151574000_2026.03.02.709196v2.45","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0150528"},{"key":"2026031009151574000_2026.03.02.709196v2.46","doi-asserted-by":"crossref","first-page":"100111","DOI":"10.1016\/j.device.2023.100111","article-title":"PyLabRobot: An open-source, hardware-agnostic interface for liquid-handling robots and accessories","volume":"1","year":"2023","journal-title":"Device"},{"key":"2026031009151574000_2026.03.02.709196v2.47","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty191"},{"key":"2026031009151574000_2026.03.02.709196v2.48","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-025-02839-x"},{"key":"2026031009151574000_2026.03.02.709196v2.49","doi-asserted-by":"publisher","DOI":"10.1111\/imr.12396"},{"key":"2026031009151574000_2026.03.02.709196v2.50","doi-asserted-by":"publisher","DOI":"10.1038\/nri3667"},{"key":"2026031009151574000_2026.03.02.709196v2.51","doi-asserted-by":"publisher","DOI":"10.1145\/1463891.1463912"},{"key":"2026031009151574000_2026.03.02.709196v2.52","doi-asserted-by":"publisher","DOI":"10.64898\/2026.03.02.709168"},{"key":"2026031009151574000_2026.03.02.709196v2.53","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1038\/s41586-025-09429-6","article-title":"One-shot design of functional protein binders with BindCraft","volume":"646","year":"2025","journal-title":"Nature"},{"key":"2026031009151574000_2026.03.02.709196v2.54","doi-asserted-by":"publisher","DOI":"10.1126\/science.add2187"},{"key":"2026031009151574000_2026.03.02.709196v2.55","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-023-06415-8"},{"key":"2026031009151574000_2026.03.02.709196v2.56","doi-asserted-by":"publisher","DOI":"10.1038\/S41586-021-03819-2"},{"key":"2026031009151574000_2026.03.02.709196v2.57","doi-asserted-by":"publisher","DOI":"10.1101\/gr.128124.111"},{"key":"2026031009151574000_2026.03.02.709196v2.58","doi-asserted-by":"publisher","DOI":"10.14806\/ej.17.1.200"},{"key":"2026031009151574000_2026.03.02.709196v2.59","doi-asserted-by":"crossref","unstructured":"Danecek, P. et al. Twelve years of SAMtools and BCFtools. Gigascience 10, (2021).","DOI":"10.1093\/gigascience\/giab008"},{"key":"2026031009151574000_2026.03.02.709196v2.60","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btw354"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.03.02.709196","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,10]],"date-time":"2026-03-10T16:15:28Z","timestamp":1773159328000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.03.02.709196"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,3]]},"references-count":60,"URL":"https:\/\/doi.org\/10.64898\/2026.03.02.709196","relation":{},"subject":[],"published":{"date-parts":[[2026,3,3]]},"subtype":"preprint"}}