Team:FSU/Poster

Poster: FSU



SPLASH
Sarah Fuller¹, Kelly Bacherman¹, Jose Miguel Serenas¹, Kajoyrie Purcell¹, Cesar Rodriguez ²

¹iGEM Student Team Members, ²iGEM Team Primary PI

Abstract:

Antibiotic resistance is an increasing global emergency that causes 2.8 millions infections in the US each year, with 750,000 worldwide deaths annually. Bacteria evolve to become resistant to certain antibiotics through selective pressures in the environment. A longitudinal study conducted on the Indian River Lagoon in Florida was used as the backbone of this project; it concluded that 88.2% of all bacterial isolates were resistant to at least one antibiotic, while resistance to erythromycin was highest at 91.6% of all isolates. The author of the study suggested that antibiotics are being polluted into Florida’s waterways via septic tanks, drainage canals, and wastewater discharge. This led us to develop a genetically engineered E. coli cell that is capable of degrading erythromycin in wastewater treatment plants. However, the engineered cell’s mechanism for degradation is the same as an antibiotic resistant bacteria; therefore, a safeguard must be put in place. A toxin/antitoxin, or TA module, kill switch was also designed to prevent the engineered cell from degrading erythromycin outside of the treatment plant. With the advice of wastewater engineers and treatment plant owners, we developed an effective solution that adds almost no cost to the consumer since the kill switch relies on compounds already found in each plant. This solution can be implemented worldwide to reduce the spread of antibiotic resistance in waterways..
Inspiration
  • We first took inspiration from the dolphins in the Indian River Lagoon. We discovered the main topic for our project when findings from a researcher by the name of Adam Schaefer at Florida Atlantic University concluded that bacteria on the dolphins' bodies were extremely resistant to antibiotics based on the Multiple Antibiotic Resistance (MAR) Index. This finding alarmed us since dolphins are a sentinel species which are animals used to detect risks to humans by providing an advance warning of danger.

  • We started to find major sources that are contributing to the problem of antibiotic resistance and we found that the agriculture, aquaculture, and wastewater treatment industries were contributing the most to this problem. We chose the wastewater treatment industry due to the scale of the amount of reclaimed water that is used which is around 1.5 billion gallons per day.

Integrated Human Practices
Our Human Practice Team’s investigation began with the dolphin study conducted on the Indian River Lagoon. The goal became to find the source of antibiotic pollution into Florida’s waters. Adam Schaefer, the author of the study, suggested that septic tanks, wastewater discharge, and drainage canals contributed to selective pressures placed on the environment. We decided to focus on wastewater treatment plants because of the established and standardized protocols.

Dolphins

  • Found a study on the Indian River Lagoon with convincing evidence of increased antibiotic resistance in Florida's waterways.
  • 91.6% of all bacterial isolates were resistant to erythromycin, a staggering statistic that led us to dig deeper into this problem.
  • Dolphins are a sentinel species, meaning they can detect warnings to humans of future health risks.
  • Adam Schaefer, the author of the study, concluded that the antibiotics are likely being introduced into the environment from septic tanks and wastewater discharge.

    • Stakeholders

  • Met with Mike Kelley, an operator of a wastewater treatment plant, to discuss a vessel for our solution. He suggested bacteria since it already used in plants and does not require any pumps, scrubbers, or bioreactors.
  • Met with Dr. Tang, a wastewater engineer at the FAMU-FSU College of Engineering, who suggested that we target antibiotics rather than antibiotic resistant genes (ARGs) since antibiotics are more prevalent and ARGs would be hard to target since they are usually inside a cell.
  • Met with Dr. Bass, a professor at FSU, who helped with our design. He helped stimulate ideas for an inducible kill switch, which led us to designing a methane-induced TA module.

    • Florida Department of Environmental Protection

  • Reached out to countless departments within the FDEP
  • Received many emails stating that it is not likely the FDEP tests for antibiotics in sewage systems
  • Some departments rerouted us to other departments, but we were unsuccessful in finding definitive antibiotic data from the FDEP, We were forced to look elsewhere.
    • Our Idea
    After months of discussion within our team and among advisors, we began work on developing an engineered E. coli cell to degrade erythromycin in wastewater treatment plants.
  • We discovered that there are companies selling chemicals and bacteria to help control the integrity and output of their treatment plant. We took inspiration from this and decided to create our own bacteria that degrades of antibiotics in treatment plants. We chose Erythromycin since it is a very common antibiotic used to treat a wide variety of bacterial infections.

  • We named our product Sewage Purification Limiting Antibiotic Spread in Habitats (SPLASH) to represent our product as a solution to help limit antibiotic the spread of antibiotic resistance through sewage and this would help the habitats surrounding the treatment plants from becoming afflicted by this problem.
    • Ere Generator
    The EreB and EreA enzyme generator produces the erythromycin esterase types I and II proteins to degrade erythromycin. To accomplish this, a constitutive promoter is used to constantly produce mRNA transcripts of the ereA and ereB genes to degrade as much erythromycin as possible in the treatment plant. The EreA and EreB enzymes work by hydrolyzing erythromycin’s lactone ring.

    Upon hydrolysis of the lactone ring, the antibiotic is rendered inactive and is no longer able to confer resistance to other microorganisms. However, it must be noted that this mechanism of action gives our engineered cell an antibiotic resistant gene. Thus, a kill switch must be implemented to prevent the continuation of resistance if the bacteria get out of the treatment plant.

    Kill Switch
    If our organism were to escape the treatment plant, it could duplicate and transfer its resistance to other organisms, prolonging the problem already plaguing communities worldwide. Therefore, a toxin/antitoxin, or TA module kill switch was designed to kill the bacteria if it were to get out. Initially, we planned to use a synthetic nutrient in the treatment plant that is not found in the water. However, Mr. Kelley told us that buying the synthetic nutrient would come out of pocket and would not be ideal for the plant owner. We decided to use a methane inducible promoter since methane is found naturally in treatment plants, but does not exist in water systems. This proved to be a challenge since we could not find a suitable promoter with methane. Therefore, we developed a solution that converts methane to methanol and methanol to formaldehyde, which induces the system to produce the toxin (ParE2) and antitoxin (ParD2). The production of ParE2 and ParD2 is dependent on the amount of formaldehyde in the treatment plant.

    In the presence of formaldehyde, both the toxin and antitoxin called are produced. They will form a heterodimer and inactivate one another, causing it to be completely harmless to the cell. In low concentrations of formaldehyde (i.e. outside the treatment plant), the ParE2 toxin will be produced, but the ParD2 antitoxin will not be produced. Therefore, the bacteria will be killed by the toxin and it will not be able to confer resistance. Even if it shares the plasmid, the receiving bacteria will produce only the toxin as well since it will be in low concentrations of formaldehyde.

    Model
    • The model we created is used to calculate how much of our enzyme we would need to remove an effective amount of erythromycin from the influent from wastewater treatment plants. The model does not account for side reactions or the influx rate of erythromycin and the rate may be reduced since there are many other substances in wastewater. We used an exponential equation based on the doubling time of E.coli to model the growth rate of bacteria.

    • The influx rate is the rate of erythromycin entering our bacteria. We decided to model the rate of erythromycin entering our bacteria since erythromycin travels inside the bacteria through active transportation because of the size and polarity of the molecule (Kojima, 2013).

    • We used the Michaelis-Menten equation to model the interaction of erythromycin with EreA and EreB. The equation is used to model the changing rate of enzyme-substrate reactions as the concentration of substrate changes (Kim, 2002). The dilution of the enzyme was accounted using a series of equations.



    • Our model was developed using MATLAB using the SimBiology package. The package provides an interface for ordinary differential equations

    • Simbiology solved 5 differential equations numerically

    • We found that 0.8L of our bacteria would have to be added to a 5000L tank to be effective.
    Build
    We did not have the opportunity to perform any experiments in the laboratory this year, but we designed a plan to build our design. First, we would have built plasmids using IDT G-blocks and the New England BioLabs Hi-Fi DNA assembly mix, which provides a painless method of assembly. We also would have designed and synthesized the regulatory g-blocks that included the BioBrick prefix, the promoter being tested, a Ribosome Binding Site, and a 25-base pair overlap of the coding sequence. We also would have designed coding sequence gBlocks which included the coding sequence, a terminator, and the BioBrick suffix. We would have used the pSB1K3 plasmid backbone.
    Test

    To test for the efficacy of the devices we designed, we would have first attempted to verify the expression of each individual generator. This includes the following:

    • EreA+EreB Generator
    • ParE2 Generator
    • Formaldehyde Inducible ParD2 Generator
    • Methane Monooxygenase Generator
    • Methanol Dehydrogenase Generator

      • The following list is a theoretical plan for testing our solution.

    • The proteins would be purified using a streptavidin affinity column as the chromatography technique. Once purified, we would have verified the presence of each protein with protein electrophoresis, analyzing the bands that occur at the known molecular weight of the individual protein.
    • Functional analysis experiments of each generator would have begun. For example, we would have grown cells containing the EreA+EreB Generator, placed them into the environment in the presence of erythromycin, and qualitatively determined if erythromycin was hydrolyzed.
    • The next step would have been to add two generators together and determine if both functions work. For example, adding the Methane Monooxygenase Generator and Methanol Dehydrogenase Generator together and analyzing if methane successfully converted to methanol and then to formaldehyde.
    • The end goal would be to put all of the generators together and analyze if erythromycin was successfully degraded and the TA module kept the bacteria alive in the presence of methane and formaldehyde.
    Proposed Implementation

    Mike Kelley, the owner of a wastewater treatment plant on the Indian River lagoon, was a key part of our implementation discussion because he is a potential consumer of our product. We met with him to discuss how our idea could be integrated into the infrastructure of the treatment plant. Along with his suggestions, we developed an implementation plan for use in any wastewater treatment plant:

    • We will not be using a synthetic nutrient for the kill switch. Instead, a methane inducible kill switch was developed to lower costs for the consumer.
    • The bacteria will be placed inside the digestor of the treatment plant since it is a methane rich environment.
    • To combat bacteria from getting into the environment, we developed a kill switch that produces a protein that is toxic to the cell in low concentrations of methane. It will also prevent other bacteria outside the plant from incorporating the dead cell’s antibiotic resistant plasmid. If another bacteria receives the plasmid via conjugation or transformation, it will begin to produce the toxin and not the antitoxin, and that bacteria will die.
    Future Directions
    • Our product currently works to degrade macrolides; a class of antibiotics that consist of a large macrocyclic lactone ring to which one or more deoxy sugars, usually cladinose and desosamine, may be attached. In the future our bacteria could be adapted to degrade other types of antibiotics which makes it versatile and able to be used in different treatment plant environments depending on the prevalent pharmaceuticals within the sewage.

    • Our product can be commercialized using the business-to-business model for revenue. We would create value by adding functionality to treatment plants by helping degrade antibiotics. This can be marketed in an environmentally-friendly way. We would be able to sell our product by weight or in a fixed package depending on the size of the treatment plant and how much of our product it would need to be effective. A market already exists for a product like ours, therefore entering the market would make us a competitive business due to our edge of providing value and functionality to treatment plants in a unique way. Since we did not have access to a lab this season, we were not able to fully field-test our product in a simulated environment. Next year if our iGEM team continues to improve upon this product we would be able to fully test and simulate its effectiveness in treatment plants. This would give our project next year a more concrete look on its functionality and interactions within treatment plant environments.
    References and Acknowledgements

    The Human Practices, Engineering Success, Modeling, and Proposed Implementation reports on our wiki have lists of all the works that were cited.

    We would like to thank Florida State University's

    • Office of the Provost
    • College of Medicine
    • Center for Undergraduate Research and Academic Engagement
    • Office of Research
    for the supported they provided this year. We would also like to acknowledge
    • Mike Kelley
    • David Montez
    • Cesar Rodriguez
    • Dr. Youneng Tang