Engineering Recombinant Plasmids For CRISPR Phage Defense & GFP Expression

• Emily Fioriello, Applied Molecular Biology & Biotechnology, University of Delaware

• Jeremy Bird, Department of Biological Sciences, University of Delaware

CRISPR-Cas systems are adaptive immune systems found in bacteria that defend against bacteriophage infection by recognizing and targeting foreign DNA. This study focuses on a Type III-A CRISPR-Cas system, which recognizes newly transcribed target RNA and promotes degradation of the corresponding DNA. This study aimed to construct recombinant plasmids using two independent molecular cloning strategies to investigate CRISPR phage defense and GFP expression. 

The first project involves testing the targeting specificity of a Type III-A CRISPR expressing plasmid by replacing the targeting spacer sequence with DNA fragment derived from T4 phage gene 12 coding sequence. After sequence verification, the CRISPR construct will be evaluated using T4 bacteriophage plaque assays performed in the presence and absence of arabinose induction to determine whether expression of the modified CRISPR system provides protection against phage infection. Arabinose induced cultures are expected to produce fewer plaques than uninduced cultures if the modified CRISPR construct provides effective phage defense. By performing CRISPR defense phage plaque assays with targeting spacers that contain gene 12 coding sequence or that are complimentary to the coding sequence, we can learn about the mechanisms of Type III-A CRISPR systems. The second project involves cloning of an inducible GFP containing expression cassette from plasmid pAJM.011 into the plasmid pBR322. The GFP construct will be examined by agarose gel electrophoresis and fluorescence microscopy to confirm successful expression of the inserted amplified gene. This new plasmid can be used as a target of the Type III-A CRISPR system to allow us to measure target DNA degradation by the CRISPR system. 

Together, these projects combine molecular cloning to investigate Type III-A CRISPR function. By developing plasmids for both phage targeting and GFP based DNA degradation, this research provides tools to study CRISPR immunity and expands our understanding of bacterial defense mechanisms.