Researcher(s)
- Morgan Wilson, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Jeremy Bird, Biological Sciences, University of Delaware
Abstract
Bacteriophages are viruses that infect bacteria. These viruses have a strong impact on the natural life cycle of bacteria. One important defense mechanism bacteria have developed to protect themselves against bacteriophage is called CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR consists of short pieces of repeats about twenty to forty base pairs that are palindromic, then spacer sequences which match up to the viral DNA. We have cloned the Type-III-A CRISPR-Cas system from S. epidermidis and made it arabinose inducible in E. coli. We chose to use the Type-III-A system because it associates with RNA polymerase and is one of the most widely used systems across prokaryotes. T4 phage are one of the viruses that infect E. coli. T4 phage which undergoes a lytic life cycle which lasts about twenty five minutes. We have looked into how the timing of transcription of the gene impacts bacterial defense against phage infection. Prior research shows that early expressed genes provide a better defense against phage infection. We have successfully cloned the non-coding strand of seven different early transcription genes, four middle transcription genes, and four late transcription genes into the pSEP_CRISPR plasmid which is arabinose inducible. Using the double layer agar assay we have studied five early genes, two middle genes, and two late genes to test the effectiveness of our cloned CRISPR targeting sequences against T4 phage. The plaque number was counted and statistical analysis to determine the impact of each gene in bacterial defense the next day. Based on our preliminary data we have determined that both the function of the gene and the timing of gene transcription has an significant impact on the level of protection of bacteria against bacteriophage conferred by a Type-III-A CRISPR-Cas system.