The inability to quickly or readily assess water samples for Escherichia coli is a major cause of serious illness around the world, especially for third world countries that cannot implement advance methods for detection. Thus, we sought to create a fast and reliable detection assay using T7 bacteriophage, highly specific to E. coli, by reducing the T7 Bacteriophage cells' lysis time while incorporating a signaling molecule gene within its genome. To do so, we engineered the T7 bacteriophage genome to included a GFP gene within the highest expressed region of the T7 bacteriophage genome followed by holin (gene 17.5) consecutively. This design ensures a fast and large output of GFP proteins after the quick lysis of the cell, thus a sensor capable of producing a visible signal upon introduction in an E.coli contaminated water sample.
Determining GFP Placement
To determine GFP placement, we chose three main areas of the T7 genome to test the insertion of GFP. The first two are clusters of nonessential genes, which are genes that have been classified previously as unnecessary for the survival of T7. We chose the 1.4, 1.5, 1.6 cluster and the 4.3, 4.5, 4.7 cluster, both located in the Class II genes. Because the phage genome has a limited amount of space to insert foreign genes, as a larger genome can become burdensome, we thought that replacing the nonessential genes with GFP would not only minimize our construct but also reveal what genes can be taken out of T7 for future engineering. However, we found that these areas have relatively low expression, which may not be ideal for our goal of creating a reporter phage. Therefore, we also tested the insertion of GFP before and after gene 10 (capsid protein) which has the highest expression in the genome due to the accumulation of promoters upstream its encoding region. We found that inserting GFP right before gene 10 optimizes its expression for strong visible signal output. Ultimately, we will be replacing the GFP gene with a chromoprotein to ensure the output of a signal readily visible and easily quantified.
Determining Lysis Gene Placement
To decrease the lysis time of the T7 Bacteriophage, we sought to move its lysis genes, namely its Holin gene, to earlier parts of its genome as their early expression would catalyze its lysis mechanism prematurely. However, we realized early on in our experiments that placing Holin in the Class I and Class II genes did not significantly decrease lysis time, as those parts of the genome generally have low expression, as also observed with GFP. We tested several locations to insert Holin relative to GFP. The main three designs we tested were inserting one or two Holins directly before gp10 and directly after GFP, and also putting Holin directly before GFP. However, we recognized that Holin should not be relocated before GFP, as this decreased the expression potential of the signaling molecule. Similarly, we found that inserting two Holins decreased lysis time too much, significantly decreasing GFP production. After comparing our three designs, we opted to include the Holin directly after the signaling molecule gene, GFP in our model. This placement ensures that GFP expression is maximized and increases the expressivity of Holin, as it falls directly before the highly expressed capsid protein. Our final design (gfp-holin-gp10), was verified to maximize GFP expression while significantly decreasing lysis time by analyzing the mutant's gene expression profile. Thus, our system optimizes a strong signal output at a quicker rate.
Heineman, R. H., Molineux, I. J., and Bull, J. J. (2005) ‘Evolutionary Robustness of an Optimal Phenotype: Re-Evolution of Lysis ina Bacteriophage Deleted for Its Lysin Gene’,Journal of MolecularEvolution, 61: 181–91
Molineux, I.J. 2006. The T7 Group, pp. 277-301. The Bacteriophages. Oxford University Press, New York.
Qadri et al. (2005) ‘Enteroteoxigenic Escherichia coli in Developing countries: Epidemiology, Microbiology, Clinical Features, Treatement, and Prevention’, American Society for Microbiology, 18: 465-483