Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species
Abstract
:1. Introduction
2. Results
2.1. T7-like Pseudomonas Phage Genomes Encode Putative RNA Polymerases, Lysozymes, and Phage-Specific Promoters
2.2. Screening Non-Toxic Phage RNA Polymerases and Their Transcriptional Activity
2.3. Phages phi15, PPPL-1, Pf-10, and 67PfluR64PP Encode Short, 17 bp Promoters
2.4. T7-like Phage Lysozymes Inhibit Transcriptional Activity of Their Corresponding Phage RNAP
2.5. T7-like Phage Lysozymes Efficiently Inhibit Phage RNAPs from Related T7-like Phages
2.6. Flow Cytometry-Based Quantitative Assessment of the phi15 Expression System
3. Discussion
4. Materials and Methods
4.1. Bacteriophage Genomes
4.2. Bacterial Manipulation
4.3. Vector Construction
4.4. Toxicity Evaluation by Growth Curve Monitoring
4.5. Fluorescence Intensity Assays
4.6. Transcription Start Site Determination with 5′-Capping-RACE
4.7. Flow Cytometry
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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P. putida Wild-Type | P. putida phi15 | P. putida phi15 (G3RQ) | |||||
FITC-A | Induced (%) | FITC-A | Induced (%) | FITC-A | Induced (%) | ||
−RNAP | +lys | 44 | 0.16 | 17,803 | 80.00 | 12,730 | 66.66 |
−RNAP | −lys | 70 | 0.12 | 15,270 | 74.74 | 32,128 | 78.50 |
+RNAP | −lys | 99 | 0.30 | 72,956 | 95.40 | 122,739 | 92.82 |
Fold induction * | 2.25 | 4.10 | 9.64 | ||||
P. aeruginosa Wild-Type | P. aeruginosa phi15 | P. aeruginosa phi15 (G3RQ) | |||||
FITC-A | Induced (%) | FITC-A | Induced (%) | FITC-A | Induced (%) | ||
−RNAP | +lys | 647 | 3.22 | 33,429 | 77.52 | 527 | 5.94 |
−RNAP | −lys | 692 | 6.18 | 7820 | 47.74 | 18,672 | 81.10 |
+RNAP | −lys | 559 | 3.22 | 245,893 | 90.98 | 68,392 | 74.58 |
Fold induction * | 0.86 | 7.36 | 129.78 |
Bacteriophage | Accession Number | Reference |
---|---|---|
Phi15 | FR823298.1 | [41] |
Pf-10 | NC_027292.1 | Unpublished |
PPPL-1 | NC_028661.1 | [42] |
67PfluR64PP | MH179478.2 | [43] |
Name | Sequence |
---|---|
TSS_TSO | ACACTCTTTCCCTACACGACGCTCTTCCGATCTrGrGrG |
TSS_outerprimer | AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT |
TSS_innerprimer | ATAGCTCTTCTAGACTACACGACGCTCTTCCGATCT |
TSS_GSP1 | TCAGTTTACCGTTGGTTGCATCACCTTCACCTTCACCACGAACAGAGAATTTGTGGCC |
TSS_GSP2 | TAGGCTCTTCTCTTCGAACAGAGAATTTGTGGCC |
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Lammens, E.-M.; Feyaerts, N.; Kerremans, A.; Boon, M.; Lavigne, R. Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species. Int. J. Mol. Sci. 2023, 24, 7175. https://doi.org/10.3390/ijms24087175
Lammens E-M, Feyaerts N, Kerremans A, Boon M, Lavigne R. Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species. International Journal of Molecular Sciences. 2023; 24(8):7175. https://doi.org/10.3390/ijms24087175
Chicago/Turabian StyleLammens, Eveline-Marie, Nathalie Feyaerts, Alison Kerremans, Maarten Boon, and Rob Lavigne. 2023. "Assessing the Orthogonality of Phage-Encoded RNA Polymerases for Tailored Synthetic Biology Applications in Pseudomonas Species" International Journal of Molecular Sciences 24, no. 8: 7175. https://doi.org/10.3390/ijms24087175