Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7
Abstract
1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Phages
2.2. Type I CRISPR gRNA Delivery Vector Component/Vector Design
2.3. Type I CRISPR gRNA Design
2.4. Type II CRISPR gRNA Design
2.5. Golden Gate Cloning
2.6. PCR
2.7. Gibson Assembly
2.8. Homologous Recombination and In Trans Complementation
2.9. Confirmation of Phage Mutants
2.10. Type I and Type II CRISPR Screening Assays
3. Results
3.1. Determination of Marker-Based (cmk and trxA) Selection Efficiency against Phage T7
3.2. Determination of the Efficiency of Type I CRISPR for Selecting against Wild-Type Phage T7
3.3. Determination of Type II CRISPR Efficiency as a Method for Engineered Phage T7 Selection
3.4. Generation of T7 Tail Fibre Mutants
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Grigonyte, A.M.; Harrison, C.; MacDonald, P.R.; Montero-Blay, A.; Tridgett, M.; Duncan, J.; Sagona, A.P.; Constantinidou, C.; Jaramillo, A.; Millard, A. Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7. Viruses 2020, 12, 193. https://doi.org/10.3390/v12020193
Grigonyte AM, Harrison C, MacDonald PR, Montero-Blay A, Tridgett M, Duncan J, Sagona AP, Constantinidou C, Jaramillo A, Millard A. Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7. Viruses. 2020; 12(2):193. https://doi.org/10.3390/v12020193
Chicago/Turabian StyleGrigonyte, Aurelija M., Christian Harrison, Paul R. MacDonald, Ariadna Montero-Blay, Matthew Tridgett, John Duncan, Antonia P. Sagona, Chrystala Constantinidou, Alfonso Jaramillo, and Andrew Millard. 2020. "Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7" Viruses 12, no. 2: 193. https://doi.org/10.3390/v12020193
APA StyleGrigonyte, A. M., Harrison, C., MacDonald, P. R., Montero-Blay, A., Tridgett, M., Duncan, J., Sagona, A. P., Constantinidou, C., Jaramillo, A., & Millard, A. (2020). Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7. Viruses, 12(2), 193. https://doi.org/10.3390/v12020193