DOI: 10.5061/dryad.pzgmsbd23
title: A fully synthetic Golden Gate assembly system for engineering a <em>Pseudomonas aeruginosa</em> phiKMV-like phage

description: Bacteriophages have applications in biotechnology, including human and
 veterinary medicine, agriculture, food safety, and biosecurity. One
 example of resurging importance is phage therapy, the use of phages to
 treat antibiotic-resistant bacterial infections. Phage therapy currently
 requires screening of environmental phages against the infecting strains
 for each individual patient, a laborious process that limits the
 development of standardized treatments. To overcome limitations of narrow
 host range inherent to many native phages, a robust genomic engineering
 platform is required, which permits rapid and dependable genome production
 and engineering for non-model phage. Here we describe an engineering
 platform for a phiKMV-like Pseudomonas aeruginosa phage, 41S1, that builds
 on previous work in the rapid assembly of small genomes through one-pot,
 High Complexity Golden Gate Assembly (HC-GGA). This system divides the
 41S1 genome into DNA fragments small enough to be conveniently synthesized
 and to avoid toxicity during DNA propagation, with all but one maintained
 in Escherichia coli. These fragments are readily assembled in a
 high-accuracy, one-pot reaction; phages can be rescued by direct
 transformation into P. aeruginosa PAO1 or E. coli 10-beta cells. We
 demonstrate the precise generation of point mutations, DNA insertions and
 deletions, and the addition of fluorescence reporter genes that are
 expressed during phage replication. All viable genotypes could be
 generated with a near 100% success rate with minimal screening. This
 system demonstrates the potential of HC-GGA for the rapid production and
 engineering of phages and provides a chassis for the development of phages
 that broadly target the opportunistic human pathogen P. aeruginosa.
description: # A fully synthetic Golden Gate assembly system for engineering a
 *Pseudomonas aeruginosa* phiKMV-like phage Dataset DOI:
 [10.5061/dryad.pzgmsbd23](https://doi.org/10.5061/dryad.pzgmsbd23) ##
 Description of the data and file structure Plasmid maps for different
 plasmids used in Golden Gate assembly of different variants of phage
 40S1and genomes of the different variants of phage 40S1 ### Files and
 variables #### File: phAK001.gb **Description:** genome of phage phAK001
 #### File: phAK002.gb **Description:** genome of phage phAK002 #### File:
 phAK003.gb **Description:** genome of phage phAK003 #### File: phAK004.gb
 **Description:** genome of phage phAK004 #### File: phAK005.gb
 **Description:** genome of phage phAK005 #### File: phAK007.gb
 **Description:** genome of phage phAK006 #### File: phAK008.gb
 **Description:** genome of phage phAK008 #### File: phAK011.gb
 **Description:** genome of phage phAK0011 #### File: phAK013.gb
 **Description:** genome of phage phAK0013 #### File: phAK009.gb
 **Description:** genome of phage phAK009 #### File: phAK006.gb
 **Description:** genome of phage phAK006 #### File: phAK014.gb
 **Description:** genome of phage phAK0014 #### File: phAK015.gb
 **Description:** genome of phage phAK0015 #### File: phAK016.gb
 **Description:** genome of phage phAK0016 #### File: phAK018.gb
 **Description:** genome of phage phAK0018 #### File: phAK012.gb
 **Description:** genome of phage phAK0012 #### File: phAK017.gb
 **Description:** genome of phage phAK0017 #### File: phAK019.gb
 **Description:** genome of phage phAK0019 #### File: phAK024.gb
 **Description:** genome of phage phAK0024 #### File: phAK020.gb
 **Description:** genome of phage phAK0020 #### File: phAK025.gb
 **Description:** genome of phage phAK0025 #### File: phAK023.gb
 **Description:** genome of phage phAK0023 #### File: phAK010.gb
 **Description:** genome of phage phAK010 #### File: phAK021.gb
 **Description:** genome of phage phAK021 #### File: F1_pTMT.gb
 **Description:** plasmid map of fragment F1 #### File: F1.2_pANSA3.gb
 **Description:** plasmid map of fragment F1.2 #### File: F1.7_pANSA3.gb
 **Description:** plasmid map of fragment 1.7 #### File: phAK022.gb
 **Description:**  genome of phage phAK022 #### File: F1.4_pANSA3.gb
 **Description:** plasmid map of fragment F1.4 #### File:
 F22_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F22
 #### File: F24_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of
 fragment F124 #### File: F25.2_pUC57-mini-BsaI-Free.gb
 **Description:** plasmid map of fragment F25.2 #### File:
 F27_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F27
 #### File: F1.3_pANSA3.gb **Description:** plasmid map of fragment F1.3
 #### File: F1.5_pANSA3.gb **Description:** plasmid map of fragment F1.5
 #### File: F4.6_pTMT.gb **Description:** plasmid map of fragment F4.6 ####
 File: F5_pTMT.gb **Description:** plasmid map of fragment F5 #### File:
 F7_pTMT.gb **Description:** plasmid map of fragment F7 #### File:
 F1.6_pANSA3.gb **Description:** plasmid map of fragment F1.6 #### File:
 F25.3_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment
 F25.3 #### File: F1.8_pANSA3.gb **Description:** plasmid map of fragment
 F1.8 #### File: F19.2_pUC57-mini-BsaI-Free.gb **Description:** plasmid map
 of fragment F19.2 #### File: F20_pUC57-mini-BsaI-Free.gb
 **Description:** plasmid map of fragment F20 #### File: F4_pTMT.gb
 **Description:** plasmid map of fragment F4 #### File: F3.2_pTMT.gb
 **Description:** plasmid map of fragment F3.2 #### File: F10_pTMT.gb
 **Description:** plasmid map of fragment F10 #### File: F4.5_pTMT.gb
 **Description:** plasmid map of fragment F4.5 #### File: F13_pTMT.gb
 **Description:** plasmid map of fragment F13 #### File: F11_pTMT.gb
 **Description:** plasmid map of fragment F11 #### File:
 F16_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F16
 #### File: F2.gb **Description:** plasmid map of fragment F2 #### File:
 F3_pTMT.gb **Description:** plasmid map of fragment F3 #### File:
 F12_pTMT.gb **Description:** plasmid map of fragment F12 #### File:
 F4.2_pTMT.gb **Description:** plasmid map of fragment F4.2 #### File:
 F19_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F19
 #### File: F15_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of
 fragment F15 #### File: F18_pUC57-mini-BsaI-Free.gb
 **Description:** plasmid map of fragment F18 #### File: F4.4_pTMT.gb
 **Description:** plasmid map of fragment F4.4 #### File:
 F14_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F14
 #### File: F23_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of
 fragment F23 #### File: F6_pTMT.gb **Description:** plasmid map of
 fragment F6 #### File: F8_pTMT.gb **Description:** plasmid map of fragment
 F8 #### File: F17_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of
 fragment F17 #### File: F21_pUC57-mini-BsaI-Free.gb
 **Description:** plasmid map of fragment F21 #### File:
 F25_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment F25
 #### File: F26_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of
 fragment F26 #### File: F26.3_pUC57-mini-BsaI-Free.gb
 **Description:** plasmid map of fragment F26.3 #### File:
 F26.2_pUC57-mini-BsaI-Free.gb **Description:** plasmid map of fragment
 F26.3 #### File: F28_pUC57-mini-BsaI-Free.gb **Description:** plasmid map
 of fragment F28 #### File: F4.3_pTMT.gb **Description:** plasmid map of
 fragment F4.3 #### File: F9_pTMT.gb **Description:** plasmid map of
 fragment F9