Preferred Name

Rachael M. St. Jacques

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Date of Graduation

Spring 2019

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Department of Biology

Advisor(s)

Steven G. Cresawn

Abstract

CRISPR arrays are a defense mechanism employed by bacteria against viral invaders. Cas proteins do the work in detecting, capturing, and integrating the viral DNA into the CRISPR array (Barrangou et al., 2007). Anti-CRISPR proteins are produced by phages, viruses that infect bacteria, to stop the bacterial host’s CRISPR-Cas complex from interrupting the phage life cycle (Bondy-Denomy, et al., 2015).

SEA-PHAGES is a course-based bacteriophage research network composed of 120 colleges and known at James Madison University as Viral Discovery. JMU uses the unsequenced Streptomyces griseus ATCC10137 as a host for bacteriophage discovery and propagation, and in this study we report the sequencing and analysis of this strain, including a search for CRISPR-Cas arrays. To determine if the S. griseus ATCC 10137 encodes CRISPR-Cas arrays, next generation sequencing and bioinformatic analyses were performed.

DNA extraction and whole genome sequencing using an Illumina MiniSeq and Oxford MinION were used to obtain sequence data from S. griseus ATCC10137. The Illumina reads were trimmed using Trimmomatic, and the Nanopore data were filtered using Filtlong. A hybrid genome assembly using the Illumina reads and Nanopore reads was generated using Unicycler, resulting in a genome assembly that was 8,576,363 bp long. To determine if CRISPR-Cas arrays were present in the genome, the assembly fasta was uploaded to CRISPRfinder. CRISPRfinder identified 3 probable CRISPR arrays, and 10 questionable regions.

Automated annotation methods Prokka and RAST were used to predict genes in the S. griseus genome, but they produced substantially different output. We therefore developed the novel bioinformatic tool, Prokkrastinator, to merge the two annotation methods. Prokkrastinator doubles as a genome browser and gene table.

To search for anti-CRISPR proteins in the genome of bacteriophage Wipeout, protein models were generated using YASARA. Of the 62.57 % of the Wipeout gene products that were not able to be modeled, 13 gene products were in the same size range as all other phage-produced anti-CRISPR proteins, 50-150 amino acids long. These 13 gene products should be further studied to determine whether or not they could be potential anti-CRISPR proteins, as they are the only proteins of the appropriate size.

Available for download on Tuesday, April 14, 2020

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