Every year, unsafe water kills more people worldwide than disasters and conflict combined. This is due to the inability of people in developing parts of the world to quickly assess the contamination level of their water sources. Most conventional methods currently used (such as the EPA method, PCR methods, etc.) are very time-consuming, take a lot of work by scientists, and are expensive. The UT Austin iGEM Team is planning to construct a quick detection apparatus using T7 bacteriophage in order to address the critical need for inexpensive, rapid, and on-site detection of biological pathogens in water. You can learn more about our project on the Description page.
Design
Our goal is to design the T7 bacteriophage so that we are able to insert a GFP reporter and achieve an optimal lysis time and burst size of the phage. This will allow the phage to quickly detect E. coli contamination in a water in a qualitative manner (glowing). T7 bacteriophage is an ideal candidate due to its small genome size and the wealth of existing scientific literature on the structure and function of this phage. This should provide many advantages over the status quo, such as: no false positives, high specificity, quicker than current techniques, inexpensive production, ease of use, and more. Our design process is described in greater detail on the Design page.
Methodology
We used computer software to create mutated genbank files based on the original wild type T7 genome. Utilizing the stochastic gene expression simulator Pinetree, we modeled these mutant genomes in order to generate protein and transcript abundance data. Scripts were created in R to take the data generated by Pinetree and use it to calculate the lysis time of the tested mutant sequence. This data was then used to determine which sequences had the most optimal lysis time and GFP production. Refer to our Experiments page for more information.
Results
Overall, our modifications of the T7 phage’s genome resulted in a successful model incorporating lysis gene movements and GFP insertion to yield a candidate phage with the optimal lysis time and GFP production. This ensures that the phage can lyse quick enough to be faster than the current detection methods, while also producing enough GFP to be clearly detectable. Refer to our Results page for more information.
Partnership
Our partnership with the TU Delft iGEM team was an integral part of our project and contributed to our understanding of T7 bacteriophage models. The TU Delft iGEM team used a model of the T7 bacteriophage that considered population dynamics via phage-host interactions. From this partnership, we learned that the concentrations of bacterial cells we aim to detect is unlikely to have many E. Coli cells lysed, therefore resulting in a lower signal. Our team also ran some simulations with the PineTree model, and the TU Delft iGEM team were able to identify the optimal locations to place the toxin they were trying to insert into the T7 Bacteriophage genome. More information can be found on the Partnership page.
Next Steps
Our next steps are taking the T7 mutations that we think have the most optimal GFP production and lysis time and testing them out in wet lab experiments. We will need to see if the GFP production and lysis time generated in lab matches those we generated in our simulations. Our primary canidate mutant involves inserting the gene for GFP before T7 bacteriophage's gene 10, and moving the holin gene between the two.
