Attempting to find the active molecule in a phage's communication system Student: Shukron Rachel Hen, Ashdod Mentor-Peer: Prof. Rotem Sorek, Weizmann Institute of Science, Rehovot ווילד טייפ שגדל בכל אחד מהמדיומים נראה כפנוטיפ רגיל, ואילו הזנים המושתקים אינם מגיבים למדיום, ולכן ניתן להבין שלא יצרנו מדיום פגום עם הזנים המושתקים אלא השתקנו גן שחשוב לליזוגניות. 1. Introduction bacteriophages (phages) are viruses of bacteria. In our research we focus on temperate phages, that can choose between two life cycles; the lytic and lysogenic. From previous work at the Sorek lab at the Weizmann Institute, it has been found that there is a difference in the infection patterns of a phage when infecting bacteria in a medium taken from previous infection- conditioned medium (CM). This phenotype implies the existence of a molecule that mediates that effect. We hypothesize that the active molecule drives the phage towards lysogeny. 2. Goal In this project we aim to find the gene in the phage that encode to the signal molecule. Figure 1 : lytic and lysogenic life cycles in a temperate phage 3. Methodology Figure 3: The dCas9 system Step 1 : we used mechanism based on the Crispr-Cas system in the phage in order create a library of silenced phage genes. We found two interesting genes- we’ll name them A and B Step 2:We created conditioned media from the strains of interest and WT, and tested their effect. Figure 4: Making CM Bacteria + phage Bacteria Step 1: growing the bacteria Step 3: clearing the Medium from all phage and bacteria Step 2: infecting part of the bacteria Control CM Phage CM 4. Results W.T presents the regular phenotype of infection in all the tested CMs. It means that we didn’t silenced the active molecule. Strains A and B present a lytic phenotype when infected in the tested CMs. It means that we probably silenced genes that are important to lysogeny. Growing and infecting in Phage and control CM from A Phage and control CM from W.T A Growing and infecting in Phage and control CM from A Phage and control CM from W.T Phage and control CM from B W.T Growing and infecting in Phage and control CM from B Phage and control CM from W.T B