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Genetic manipulation of Bacteriophage T4 utilizing the CRISPR-Cas13b system

ABSTRACT: T4 genome editing using CRISPR-Cas13b

PROVIDER: PRJEB81510 | ENA |

REPOSITORIES: ENA

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Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.

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Setting up a better infection: Overexpression of the early bacteriophage T4 gene motB during infection results in a more favorable tRNA pool for the phage

Project description:Purpose: We investigated the effect of the T4 MotB protein on E. coli gene expression Method: E. coli BL21 (DE3) containing either pNW129 or pNW129-MotB were grown to early log phase (OD600 ~ 0.3) then induced with 0.2% arabinose for 20 minutes. T4 phage added to the culture at MOI10. Cells were then harvested at 0, 5, and 10 min time points and total RNA was isolated. The cDNA library was prepared using a modified RNATagSeq workflow as previously described (Shishkin, A.A. et al. 2015 Nat Methods). Optimum fragmentation of the total RNA samples in this library was determined to be 3 min at 94C in FastAP buffer (Thermo Fischer Scientific). The cDNA Library was run on a Bioanalyzer using the Agilent High Sensitivity DNA Kit to evaluate the quality of the library. The concentration of the cDNA library was determined by qPCR using the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA, USA) and CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Sequencing was performed by the NIDDK Genomics Core facility using a MiSeq system with the single-end 50 bp Sequencing Kit (Illumina, San Diego, CA, USA). RNA-seq data was processed as previously described using E. coli str. K-12 substr. MG1655 (NC_000913.3) as the reference genome. Differential expression between conditions was represented as a fold change, and genes with both a fold change ≥2 or ≤ 0.5 and adjusted p value ≤ 0.05 were considered significant. Results: RNA-seq data revealed that the expression of 542 E. coli genes were significantly changed after motB expression. The expression of 704 and 706 E. coli genes were changed T4 5 min and 10 min post infection after MotB expression, respectively. After 5 min T4 infection, 34 T4 genes were significantly changed. Conclusion: T4 MotB modifies the pool of host tRNAs, creating a better infection for T4

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