This readme.txt file was generated on 2024-02-12 by Vincent Noireaux Recommended citation for the data: Levrier, Antoine; Nash, Bruce; Bowden, Steven; Lindner, Ariel; Noireaux, Vincent; Karpathakis, Ioannis. (2024). Data supporting "PHEIGES, all-cell-free phage synthesis and selection from engineered genomes". Retrieved from the Data Repository for the University of Minnesota. https://doi.org/10.13020/0CDV-Q695. ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset: Data supporting "PHEIGES, all-cell-free phage synthesis and selection from engineered genomes" 2. Author Information Author Contact: Vincent Noireaux (noireaux@umn.edu) Name: Antoine Levrier Institution: (1) School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA, (2) Université Paris Cité, INSERM U1284, Center for Research and Interdisciplinarity, F-75006 Paris, France Email: alevrier@umn.edu ORCID: 0009-0002-1649-8328 Name: Ioannis Karpathakis Institution: (1) School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA, (3) Facultatea de Biotehnologii, USAMV Bucuresti, Sector 1, Cod 011464, Bucureşti, Romania Email: iannis.karpathakis@gmail.com ORCID: Name: Bruce Nash Institution: (4) DNA Learning Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA Email: ericbrucenash@gmail.com ORCID: Name: Steven Bowden Institution: (5) Department of Food Science and Nutrition, University of Minnesota, Saint Paul, MN 55108, USA Email: sbowden@umn.edu ORCID: Name: Ariel Lindner Institution: (2) Université Paris Cité, INSERM U1284, Center for Research and Interdisciplinarity, F-75006 Paris, France Email: ariel.lindner@cri-paris.org ORCID: 0000-0001-5015-6511 Name: Vincent Noireaux Institution: (1) School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA Email: noireaux@umn.edu ORCID: 0000-0002-5213-273X 3. Date published or finalized for release: 2024-01-29 4. Date of data collection (single date, range, approximate date): 2022-08-30 to 2023-12-01 5. Geographic location of data collection (where was data collected?): N/A 6. Information about funding sources that supported the collection of the data: National Science Foundation CBET FMRG 2228971 Fulbright U.S. Student Program, which is sponsored by the U.S. Department of State, the Romanian-U.S. Fulbright Commission, and the Franco-American Fulbright Commission Bettencourt Schueller Foundation 7. Overview of the data (abstract): Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome. -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: CC0 1.0 Universal (http://creativecommons.org/publicdomain/zero/1.0/) 2. Links to publications that cite or use the data: 3. Was data derived from another source? No If yes, list source(s): 4. Terms of Use: Data Repository for the U of Minnesota (DRUM) By using these files, users agree to the Terms of Use. https://conservancy.umn.edu/pages/drum/policies/#terms-of-use --------------------- DATA & FILE OVERVIEW --------------------- File List Filename: movie raw images.zip Short description: Supplementary Movie: Raw microscopy acquisitions for E. coli B infection by T7-mC-gp10 timelapse. Filename: compil raw data.zip Short description: Figure 6.c : Supplementary Data micrographs for E. coli rfaC infected by T7-compile Filename: NGS_illumina.zip Short description: Figure 5.c : Raw NGS data for mutagenic PCR tip of the tail fiber fragments. Filename:raw-sequencing.zip Short description: All the raw sequencing obtained for the 26 fully sequenced phages described in the paper as well as the alignment files for each phage genome. 2. Relationship between files: none -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: microscopy (phase contrast, fluorescence), next generation sequencing, nanowire sequencing, fluorometry 2. Methods for processing the data: Python (graphs), Benchling (sequencing) 3. Instrument- or software-specific information needed to interpret the data: Python, Benchling 4. Standards and calibration information, if appropriate: NA 5. Environmental/experimental conditions: aerobic conditions, temperature 29-37C 6. Describe any quality-assurance procedures performed on the data: multiple repeats of the experiments were made 7. People involved with sample collection, processing, analysis and/or submission: all the authors ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: movie raw images.zip ----------------------------------------- Directory Structure: movie raw images.zip |_______BF |____bright field images (T7-mCherry cassette inserted after gp10 into T7 genome. Images that show infection of E. Coli B cells by the gp10-T7-mCherry phage) |_______TexRed |____ fluorescence images (T7-mCherry cassette inserted after gp10 into T7 genome. Images that show infection of E. Coli B cells by the gp10-T7-mCherry phage) ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: compil raw data.zip ----------------------------------------- |_______BF |____bright field images (mini-reLPS-mCherry: phage T7 mini with tail fibers specific to ReLPS and expressing mCherry) |_______TexRed |____ fluorescence images (mini-reLPS-mCherry: phage T7 mini with tail fibers specific to ReLPS and expressing mCherry) ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: NGS_illumina.zip ----------------------------------------- Directory Structure: NGS_illumina.zip |_______NGS_illumina |____NGS_illumina |_____Noireaux_Project_003-fastqs |_____E0 (fragment E0 as described in text), S1: sample1 (used by sequencing facility), R1 (read 1). Same for E1, E2, E3, E4. ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: raw-sequencing.zip ----------------------------------------- Directory Structure: raw-sequencing |_____alignments for assembly |_____each file name corresponds to the sequence of one phage genome reassembled from the data in the 'raw data' folder. The names and description of these T7 phages are in the manuscript. |_____raw data |_____each folder name corresponds to one phage with its raw sequencing data. In each folder, the sequencing data of each of the 4 fragments (A, B, C, D) that were sequenced for each phage and that enable getting the full sequence of each phage shown in the folder 'alignments for assembly).