The inability to quickly and reliably assess the safety of an unknown water source led to us focusing on engineering a T7 Bacteriophage as a sensor for microbial contamination in water. However, to truly gauge the requirements, applications, and need of such a reporter phage beyond our laboratory environment, we reached out to facilities that face water contamination as a critical obstacle. Specifically, we reached out to potential stakeholders who are looking for faster and less expensive alternatives to current bacterial detection methods. As such, we reached out to The Texas Beach Watch, U.S Military, and miniPCR.
Environmental Water Testing
The surveillance of beaches, rivers, and pools by testing facilities and programs ensures the public's safety from recreational water illness(RWI) and other related illnesses. To better understand the issues currently faced in the industry and their potential need for a new detection device, we spoke to Jason Pinchback and Lucy Flores, project managers of the Texas Beach Watch, about their experiences with current bacterial contamination detection methods and their thoughts on our proposed device.
During our meeting, Mr. Pinchbank emphasized the need for a faster detection method so that quick decisions can be made for public health safety. The current methods employed take an average of 24 hours to analyze a sample of recreational water for bacterial contamination, such as the highly used industry-standard IDEXX method (Schang et al.). Ideally, Pinchback and Flores stated, a new product "should have a quicker turnaround from 24 hours to 2-3 hours" with "quantifiable results". Additionally, they stated that "a new product with a cheap cost would always be ideal,", as this would allow them to invest and expand their opportunities to improve the quality of recreational water and visitors' experience. Mr. PinchBack and Mrs. Flores also highlighted the importance of the specific detection of bacteria dangerous to humans. Most notably, he highlighted the detection of the pathogenic bacterial strains commonly found in human feces, as these bacterial strains are the ones that most commonly cause human illness, such as RWI. Additionally, such specificity would deter away from unreliable results that may arise from current testing methods, such as the interference by natural minerals within tested water samples on a PCR based assay. Finally, Mr. Pinchback emphasized the impact a fast reporting device could have on the industry, stating that large data collection can "influence policies related to the treatment of [recreational] water." Overall, the Texas Beach Watch emphasized the potential need for a new device, and they also gave some guidelines about how a new device should:
- Provide quantifiable results within a short period of time
- Should be inexpensive
- Should be highly specific to bacterial strains dangerous to humans
The ability to quickly assess indigenous water quality is an important property described by the U.S. Department of Defense. Having talked to the Department of Defense, we learned that soldiers within the U.S. Army require up to 15 L of water per day to sustain hydration levels properly. To meet this requirement, soldiers rely heavily on the clean water they physically carry. However, soldiers must obtain water from indigenous sources when they are critically low in supplies or during other emergencies. In such cases, soldiers must send a water sample to a centralized laboratory facility to evaluate its contamination and safety. While the soldiers wait for the results, they can become dehydrated. In addition, if the soldiers decide to drink the untested water, they may suffer from acute or chronic health effects. Thus, the U.S. Army emphasized the need for an accurate and useful device to quickly assess water quality for the wellness of our troops.
With this in mind, they notified us that such a device should meet specific requirements for it to be successful within their scope. One major aspect is that the device must be small and lightweight. This requirement secures the carriers from being burdened with extra luggage, which could hinder their abilities. Additionally, the device must be low powered and usable onsite. This again should prevent the need to carry excess materials needed to use the device or even send the sample to a station where the device needs to be tailored. Finally, they emphasize the need for the device to be easy-to-use with rapid results. In summary, the U.S. Department of Defense required that the device is:
- Small, lightweight, and low powered
- usable onsite and output rapid results
- easily used with results that are easy to interpret
The ability to extend our assay outside the lab was a crucial component of our original design, and it was emphasized by our previous contacts. This design aspect ensures the ability to test and assess a water sample directly and quickly to ensure the user's health. To determine the ability of our design as an on-site tool, we contacted Sebastian Kraves, co-founder of miniPCR, a company specializing in creating products that are easily accessible and portable.
During this meeting, we addressed the importance of creating an ideal 37°C environment so that we can promote optimal phage infection and increase the reliability and effectiveness of our assay. With the help of Mr. Kraves, we learned that this requirement could be easily met with a small, portable heat-bath. These devices are low-powered, quick, and easy-to-use; thus, they ought to be considered for a final product. Additionally, with the help of miniPCR, we evaluated the ability to include the quantification of E. coli within our assay. Specifically, since our final signal is colorimetric, we discussed the ability to use a spectrophotometer to measure absorbance and relate that to bacterial E. coli concentration. Spectrophotometers, however, are not easily portable, nor are they easily accessible. Spectrophotometry would require some knowledge regarding lab procedures, creating concentration curves, etc.—both of which were advised against by the military and recreational facilities. With Mr. Kraves, we were able to talk through our options and determined that there would be a trade-off between on-site quantification and expertise, as most instruments would require a minimum knowledge of use..
Integrating Human Practices
With the feedback we received from both stakeholders, there were technical requirements that our current proposal met. Notably, using a T7 Bacteriophage is highly specific to Escherichia coli, a common water pollutant typically derived from human and animal feces. Additionally, using a phage assay would be inexpensive to make and would also eliminate the need for excessive lab equipment, thus decreasing overall cost while also requiring low power.
However, these stakeholders proposed technical requirements that our current design did not include, which ultimately influenced our project's decisions. One suggestion we are exploring is to substitute the Green Fluorescent Protein(GFP) in our current model to a chromoprotein when we test our device in the wet lab during the second year of this project. As characterized by the Department of Defense, the device should be small and lightweight; thus, by using a chromoprotein, we would avoid the need to make space within a kit to include substrates or some sort of black light to visualize GFP. This exclusion also means the device would be less expensive for both stakeholders, as there would be no extra need to synthesize the substrate or the need to purchase black lights. Additionally, using a chromoprotein as our output signal makes results super easy to interpret, since the only possible outcomes would be the expression of color if bacteria is present and no expression of color if no bacteria is present.
The need for fast outputs by both stakeholders was another factor within our design that we had to optimize. Typically, a T7 bacteriophage's infection cycle is around 25 minutes at 30°C. This means that our assay would take roughly 25 minutes to generate a signal if testing contaminated water samples. We felt the need to further decrease this detection time to guarantee a faster and more reliable device. We approached this issue by refactoring the T7 Bacteriophage genome by moving lysis genes to ensure a quicker lysis time; this ensures that the bacteriophages can replicate and spread throughout the sample faster to produce more GFP. Additionally, we placed the GFP gene within a highly expressed T7 genome region to create a stronger signal upon lysis. The speed of this assay ensures a quick assessment of water quality and the ability to gather more data for faster decisions.
Finally, after contacting these industries, we were met with two major responsibilities for a physical product: on-site detection and quantifying output. Specifically, we first needed to create a 37℃ environment for optimal phage infection, which would therefore result in faster signal production. In order to do so, we focused on including a portable heat-bath, as suggested by miniPCR. This design aspect would not only optimize our assay, but it would also expand the effectiveness of testing different water sources across varying environments. Additionally, as described by miniPCR, these devices are low-power, quick, and easy-to-use, which fit the requirements idealized by The Texas Beach Watch and the Military.
Secondly, as per recommendations from recreational water facilities, we needed a product that would output an exact number of bacteria present in the water sample. This requirement was more difficult; since our final signal is colorimetric, a spectrophotometer would be required to quantify output. Using a spectrophotometer, we can determine the amount of GFP within a water sample and back-calculate it to determine the number of bacteria cells detected. Doing this beforehand and providing the data could allow the user just to read the sample using a spectrophotometer and quickly assess the sample. However, spectrophotometers are hard to access, require prior knowledge for use, and are not easily portable. Additionally, the quantification of bacteria was only emphasized by The Texas Beach Watch, while the Department of Defense merely wanted visual conformation. To compromise, we decided to exclude a spectrometer to avoid an increased expense and product size. However, since The Texas Beach Watch and other stakeholders needing quantification may already have a spectrophotometer within their lab, we could include a data sheet correlating spectrophotometry readings to a set of bacterial cell concentration. This data sheet would only require these stakeholders to obtain samples, add our phage, and quickly measure concentration using their lab spectrophotometer. While this would require the transfer of samples from the source to in lab, it would still greatly reduce the time needed to collect data.
By talking to these stakeholders, we confirmed the benefit our device could bring to potential stakeholders. We were also able to improve our device to meet current needs we were not aware of. Ultimately, we are creating an inexpensive and easy-to-use assay to determine the presence of E. coli contamination within water samples, with both on-site and in-lab potentials.
Schang et al. (2012)‘Evaluation of Techniques for Measuring Microbial Hazards in Bathing Waters: A Comparative Study’. PLOS ONE, 11(5), e0155848.