Working in a wet lab may not seem to have much involvement with engineering. However, in order to be successful, our wet lab process follows the engineering design cycle of research, imagine, design, build, test, learn, and improve. Hence, the process of phage therapy, or more specifically biopanning and ELISA, requires incorporation of engineering principles to a certain extent.
The first and foremost step to be able to successfully work efficiently in the wet lab is to understand and know what it is that we are doing. To understand, one must research! As high school students, it would be impossible for us to be able to process scientific protocols and analysis, let alone in a limited amount of time, without having to research. All wet lab team members spent hours browsing numerous scientific papers in order to acquire a basic understanding of the process. We researched more on how biopanning is performed, ELISA is analyzed, and gene sequencing is utilized with all focus surrounding our target bacteria.
Before starting the wet lab, we had to imagine how we could get sequences of the target protein, or receptor binding protein, since that is the final result that we wish to achieve from the lab. It is crucial for us to imagine how each part of the process will ultimately form the big picture. Starting from plotting a growth curve of S. aureus, conducting biopanning of the KM13 phages, precipitating the phages, analyzing ELISA, and finally sequencing the gene sequence of the receptor binding domain.
In a more practical manner, due to our work schedule in the wet lab overlapping with our school days, the wet lab team had to alternate different members to go work at the wet lab on each day. Each day working at the wet lab was filled with meticulous details and packed with immense progress. With a different member going to the wet lab each day and having to continue the previous day’s work, a lot of coordination and imagination had to take place. Every member had to report what they did along with sending the lab notes in order for the next person to understand how to continue work on the next day. Since we did not experience the work ourselves first-hand, we had to imagine the different steps that took place.
Ideally, biopanning should be completed three times for three weeks with a week of ELISA after that. However, since our team had an extremely limited amount of time to work in the wet lab due to COVID-19 disrupting our initial plan, we were only able to complete biopanning once for one week along with three days of ELISA. Thus, we had to make changes to the protocol design and schedule in order to finish the work in time. In other words, we had to design a new schedule to accommodate the limited time we have.
The biopanning process is made up of many separate steps that build up on each other. We had to start with growing the S. aureus so that we are able to use it during its log curve or the phase where its growth is favorable for the experiment. This is then later used in the biopanning process for the KM13 phage to bind to the bacteria. Once achieved, ELISA is conducted to analyze its binding affinity. Finally, the selected phage is sent to be sequenced so that the binding domain can be further analyzed. You see, all these steps are all equally important since the next step could not be done without the previous one completed. They all build up on each other to create the final result.
A more specific example is the ELISA process itself. To simply explain, this procedure consists of a series of steps building on each other. We had to bound the phages onto the S. aureus. After that, we added an anti-KM13 antibody as our primary antibody, HRP conjugate as our secondary antibody, and finally the TMB substrate. The anti-KM13 antibody binds to the phages with the KM13 phage attached. The HRP (horseradish peroxidase) conjugate binds to the anti-KM13 phage to show us the level of affinity present. Finally, the TMB substrate plays a role in changing the solution to a blue color. This blue color will then be reacted in an oxidation reaction to change into a yellow color, indicating that a phage has successfully been bound to S. aureus.
In the wet lab, we conducted the catalase test which is a biochemical test that detects if the organism produces the catalase enzyme. We tested it on S. aureus which is a catalase-positive organism. This meant that when we dropped in hydrogen peroxide onto S. aureus, catalase would hydrolyze the hydrogen peroxide into water and gaseous oxygen, resulting in a reaction of gas bubbles. This test ensures if the organism that we are working with is purely that organism and not contaminated with any other species.
Of course, the wet lab process has definitely made us learn more than we could ever imagine. From simple procedures such as how to use a pipette to more complex protocols of washing phages, we all have taken snippets of new knowledge. Along the process, our lab instructor continued to explain how to conduct the different processes, why they are done, and where they fit in the final result.
One of the biggest learning experiences was when we conducted biopanning. Since it is quite a complex process, there were times when the biopanning results failed. Once, there were no visible S. aureus bacteria that grew while another time, there were too many colonies on the plate! Hence, we learned from our mistakes until we successfully completed the biopanning results through this learning curve.
The wet lab can definitely be improved in many ways. Due to COVID-19, we could not receive the bacteria we wanted to use which was S. pyogenes; instead we had to use S. aureus. Also, our results could be more accurate and reliable if we had time to repeat biopanning three times instead of just once. In terms of improvements whilst working in the lab, we can always minimize the errors that we made. For instance, we spilled a test tube of a specific phage, making it unable to be used anymore. Thus, these mistakes can be minimized and our methodology can be improved to ensure more reliable results.