Team:Utah State/Design

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Design of Devices for Phage Detection and Control

We have designed an experiment leveraging the natural biology of the problematic lactococcal P335 species phage Φ31 to help detect and fight against Φ31 infections of Lactococcus lactis during cheese fermentations. Our designed experiment investigates the use of two Φ31 promoters, a divergent promoter (p2001) located within the genetic switch region of the phage genome and a middle promoter (p1997), for detection and a triggered defense system. Both promoter systems investigated in this experiment have previously been determined to initiate transcription and subsequent expression in the initial stages of infection, specifically in the rightward direction of p2001, and only during phage infection. Making them excellent candidates for use in a detection and defense triggered system.

Our Φ31 phage detection system was designed utilizing these promoters and the reporter genes superfolder green fluorescent protein (sfGFP) and mCherry (a red fluorescent protein, RFP), inserted within the lactic acid bacteria (LAB) expression vector pTRKH2. In this system, transcription and subsequent expression of the fluorescent reporter proteins would only be initiated during Φ31 phage infection of L. lactis, from the designed construct within the cell, and allow for qualitative and quantitative detection by observing and measuring fluorescence, respectively. This detection system provides a rapid detection method that could be implemented within dairy fermentation facilities, allowing for timely monitoring of appearance and presence of the Φ31 phage.

The Φ31 phage triggered defense system was designed using the same promoter concept, but with expression of lalR, a three-gene restriction cassette (depicted as a single gene and enzyme for simplicity), after initial phage infection. Expression of this restriction cassette results in digestion of the bacterial chromosomal DNA, and subsequently in cell death. Because transcription from the Φ31 promoters is only expressed during Φ31 infection, cell death would only be induced during an active infection. This system is designed to provide an effective defense against Φ31 infection of L. lactis industrial dairy fermentations that results in cell death of infected cells and prevents propagation of the Φ31 phage, allowing for minimal hindrance of industrial fermentations during Φ31 infections.

Additionally, this experiment was designed purposefully incorporating the divergent p2001 promoter because of its conservation and high sequence similarity with the other lytic members of the lactococcal P335 species phage. With the assumption that expression can potentially be induced from this promoter by other lytic members, allowing for broader detection and defense against one of the main phage species that infect and ruin industrial cheese fermentations.

Fluorescent detection of Φ31: Upon injection of the Φ31 chromosome expression from the p2001 rightward promoter or the p1997 promoter is activated in our designed construct, resulting in production of green fluorescent protein.

Triggered defense of Φ31: Upon injection of the Φ31 chromosome expression from the p2001 rightward promoter or the p1997 promoter is activated in our designed construct, resulting in expression of the three-gene restriction cassette lalR. Subsequently leading to restriction digestion of the host chromosome, and cell death.