Presented by Team UFlorida 2020
S. Doshi¹, D. DuPuis¹, C. Ferguson¹, S. Golden¹, J. Mallinger¹, D. Murcia¹, B. Stone¹, R. Thelwell¹, C. Wilson¹, J. York¹, Chris Reisch²,
¹iGEM Student Team Members, ²iGEM Team Primary PI
Abstract
Team Florida 2020 takes inspiration from their 2019 project to improve and apply the novel biosensor technology that detects and records phosphorous levels to real-world ecological implications. This project couples the Synthetic Cellular Recorders Integrating Biological Events (SCRIBE) system with the naturally occurring PhoB-PhoR system in E.coli. In response to phosphorus, SCRIBE utilizes a reverse transcriptase enzyme to produce single stranded DNA which can be incorporated into the host-genome during DNA replication using the Beta Recombinase protein which results in a mutation within the bacterial chromosome. This system can be utilized to measure the amount of phosphorus in a body of water by modeling the fraction of cells that gain the mutation per generation. In the absence of a lab, the UF iGEM team seeks to model this hypothetical biosensor as part one of a two-part project.
Team:UFlorida/Poster
Aquatic Phosphorus Detection Using SCRIBE
Introduction
Team UFlorida has taken it upon themselves to develop a solution to tackle harmful algal blooms at the source. The link between increased phosphorus levels and harmful algal blooms has been extensively cited in literature. These blooms have also cost the state of Florida $14.5 million in clean-up cost. Thus, it is pertinent to the Florida economy and ecosystem to create a phosphorus detection method that can be used in our waterways. To do this, Team UFlorida has developed a biosensor that can detect above safe levels of phosphorous in bodies of water. The goals of our project throughout the year were as follows:
2. To stimulate this biosensor in MATLAB and develop an experimental plan to build and test this biosensor in the lab in 2021 as part two of our two-part project
1. To develop a solution to tackle Florida's harmful algal blooms at the source by developing a biosensor
2. To stimulate this biosensor in MATLAB and develop an experimental plan to build and test this biosensor in the lab in 2021 as part two of our two-part project
Problem
ECONOMIC EFFECTS OF HARMFUL ALGAL BLOOMS: Harmful algal blooms devestate Florida's economy by disrupting tourism and fishing industries, as well as cause an estimated 2.2 million dollars yearly in eutrophication damages
EUTROPHICATION: Eutrophication occurs when a body of water becomes overly enriched with nutrients, predominantly due to human industrial activity. Excess nutrients cause overgrowth of algae that diminishes the oxygen supply to other organisms; therefore, threatening the biodiversity of the ecosystem.
RED TIDE: Red tide algal blooms and other harmful algal blooms often lead to massacre scenes, leaving hundreds of deceased fish blanketing beaches and riverbanks. As a result, the state of Florida has spent 14.5 million dollars in clean up costs. There is sufficient evidence supporting that fertilizer run-off from industrial agriculture sites is a major cause of the harmful algal blooms responsible for red tide, with a main ingredient in fertilizer being phosphorus.
Inspiration: Team Florida's 2019 Project and the PhoBR Operon
To protect our waterways and wetland ecosystems from this devastation, we have taken on the challenge of creating a biosensor that can detect above-safe levels of phosphorus in samples of water. This biosensor would allow ecologists to make informed decisions about the impacted waterways.
More specifically, we have taken inspiration from our 2019 project to improve and apply the SCRIBE system by coupling it with the naturally occurring PhoB-PhoR phosphate sequestering system in E.coli.
The PhoBR genes work as follows:
Low phosphorus conditions: PhoR is turned on --> phosphorylates PhoB --> activates promoter PhoA and E.coli takes in phosphate
High phosphorous conditions: The system is turned off (PhoR not activated)
PhoBR image reference: Uluseker, et. all. Mit Press Journals, 2017
More specifically, we have taken inspiration from our 2019 project to improve and apply the SCRIBE system by coupling it with the naturally occurring PhoB-PhoR phosphate sequestering system in E.coli.
Low phosphorus conditions: PhoR is turned on --> phosphorylates PhoB --> activates promoter PhoA and E.coli takes in phosphate
High phosphorous conditions: The system is turned off (PhoR not activated)
PhoBR image reference: Uluseker, et. all. Mit Press Journals, 2017
Our Genetic Circuit
Team Florida has taken the PhoBR system and added a genetic inverter to enable it to respond to high phosphorus levels. The genetic inverter consists of Tetr and pTet which are placed after promoter PhoA, and before SCRIBE.
Low phosphorus conditions: pPhoA is activated --> Tetr translated --> Tetr binds and represses the pTet promoter --> SCRIBE is not transcribed.
Low phosphorus conditions: pPhoA is activated --> Tetr translated --> Tetr binds and represses the pTet promoter --> SCRIBE is not transcribed.
High phosphorous conditions: pTet promoter remains active --> SCRIBE is transcribed and a mutation results in the bacterial chromosome leading to the quantification of phosphorus in a given environment.
SCRIBE
SCRIBE consists of the following: Inducible promoter, msr, msd(rpoB), RBS, Reverse Transcriptase, RBS, Beta Recombinase Protein, and Terminator.
This mutation allows the cells to be resistant to rifampicin. In order to quantify the amount of phosphorus in a given body of water, the fraction of cells that gain the mutation per generation is measured by plating the cells on rifampicin. In the absence of a lab, we sought out to evaluate these outcomes by using mathematical modelling in MATLAB.
The promoter, when induced by phosphorus, begins transcription of the SCRIBE system. Following translation, the reverse transcriptase turns the msr/msd region from single stranded mRNA to single stranded DNA (ssDNA), with the target rpoB mutation. The Beta Recombinase binds to the ssDNA strand, containing the mutation. Then, the Beta Recombinase inserts it into the Okazaki fragment into the bacterial chromosome using site specific homologous recombination during chromosomal replication.
This mutation allows the cells to be resistant to rifampicin. In order to quantify the amount of phosphorus in a given body of water, the fraction of cells that gain the mutation per generation is measured by plating the cells on rifampicin. In the absence of a lab, we sought out to evaluate these outcomes by using mathematical modelling in MATLAB.
Why SCRIBE
- Most quantification methods utilize fluorescence proteins, like GFP. The disadvantage is that they only provide a temporary modification, as they are proteins that degrade with time.
- By making changes to a bacterial chromosome, SCRIBE would allow for the cell’s environment to be permanently recorded overtime.
- A fluorescence protein would only be expressed as a result of the promoter being turned on (short influx of phosphorus --> the protein expression halted)
Human Practices and Integrated Human Practices
Florida Sea Grant: Collaborated and contextualized our project with experts in the field from the UF Florida Sea Grant Team through a productive discussion surrounding:
Language Accessibility: In an effort to increase accessibility of our project, we consulted UF language professors in translating our project summary into several languages such as:
Ethics Professors: In order to ensure the safety and ethics of our project, we consulted two environmental ethics professors at UF, Dr. Anna Peterson and Dr. Jeffrey Burkhardt. These ethics professors introduced us to a popular concept in environmental ethics called the precautionary principle, which is based on four central tenets.
Environmental Protection Agency: To further explore how to safely implement our biosensor, as well as gain more insight on current phosphorus detection mechanisms, we met with EPA representative Daryll Joyner. Mr. Joyner, a Program Administrator for Water Quality Standards at the Florida EPA, informed us of the potential hazards of releasing E. coli into waterways. He helped us devise a way to safely deploy our biosensor without posing these risks, by utilizing a capsule-like membrane to enclose our sensor. This would prevent the sensor from interfering with EPA E. coli readings and from adding a potentially harmful pathogen into our waterways.
- Current phosphorus detection technology
- The environmental and economic impact of eutrophication
- Importance of biosensing in prevention of eutrophication
Language Accessibility: In an effort to increase accessibility of our project, we consulted UF language professors in translating our project summary into several languages such as:
- Spanish
- Mandarin
- Arabic
- Japanese
Ethics Professors: In order to ensure the safety and ethics of our project, we consulted two environmental ethics professors at UF, Dr. Anna Peterson and Dr. Jeffrey Burkhardt. These ethics professors introduced us to a popular concept in environmental ethics called the precautionary principle, which is based on four central tenets.
- Taking preventive action in the face of uncertainty.
- Shifting the burden of proof to the proponents.
- Exploring a wide range of alternatives to possibly harmful actions.
- Increasing public participation in decision making.
Environmental Protection Agency: To further explore how to safely implement our biosensor, as well as gain more insight on current phosphorus detection mechanisms, we met with EPA representative Daryll Joyner. Mr. Joyner, a Program Administrator for Water Quality Standards at the Florida EPA, informed us of the potential hazards of releasing E. coli into waterways. He helped us devise a way to safely deploy our biosensor without posing these risks, by utilizing a capsule-like membrane to enclose our sensor. This would prevent the sensor from interfering with EPA E. coli readings and from adding a potentially harmful pathogen into our waterways.
Science Communication
Bite-Sized Science: Our easily accessible Bite-Sized Science webinar allowed us to share the importance of our project and its goals to members people all across Florida. Through Florida Sea Grant, we were able to do a virtual presentation on the details and objectives of our project. We were able to connect with 34 attendees across several countries throughout the state. In a post-presentation survey, 75% of attendees had reported to have learned a lot. This effort allowed us to inform members of our community with our project in a safe and effective way despite the current pandemic.
Image from UF IFAS Florida Sea Grant Program
FSU Collab: Teaming up with Florida State University allowed us to create a collaborative video showcasing the iGEM competition and both universities’ past and current projects. Each team’s members contributed in gathering footage, providing voiceovers, and editing the video. The completed video was then distributed to high schools in Florida. A representative from both colleges presented the video at College Academy at Broward College and held a Q&A session afterwards where students were able to learn more about iGEM and the importance of pursuing research.
Proposed Implementation
In late October, team Florida was able to meet with Daryll Joyner, the Program Administrator for the Water Quality Standards Development of the Florida Environmental Protection Agency, in order to discuss proposed end users of our biosensor, unforeseen upcoming challenges of distribution, and accessibility of the product.
- In order to easily collect our biosensor after a period of time, and to reduce the likeness of interfering with EPA E. coli readings, team Florida proposes that our bionsensor be put in a capsule-like membrane that separates it from the aquatic environment.
- This “cells in a tube” idea will allow scientists to attach a GPS tracking device or a buoy to retrieve the tubes to collect data.
- We will test our parameters on multiple flow cytometers
- By putting our sensor in a small membrane and releasing it into a waterway, we reduce the likelihood of any malfeasance affecting detection.
Next Steps (Part Two)
Expand our Model: Capture the inhibitory effect of high levels of inorganic phosphate on our system through a proxy constant/function derived from experimental data regarding the aforementioned complex regulatory system involved in phosphate starvation
PhoB-PhoR Pathway
Genetic inverter
SCRIBE
Our model presupposes two major assumptions:
- All SCRIBE dynamics are functionally identical and reliant only on the concentrations of pTet an TetR molecules
- That the PhoB-PhoR phosphate starvation system can reliably be utilized to track phosphorus levels within a cellular system
Limitations:
500uM Pi
Experiment: Build and transform our PhoBR and SCRIBE plasmids into competent E.coli cells using PCR, gel electrophoresis, cloning CPEC, transformation and plating, and colony PCR
Test our system: Evaluate outcomes by computing the fraction of cells that gain the mutation per generation. Induce cells with phosphorous and plate with and without rifampicin. This will give the total cell count, as well as the number of cells with rifampicin resistance. Dividing the number of rifampicin resistant with total number gives the fraction of cells that have the mutation.
We Could Not Have Done It Without You!
Donors
Dr. David Norton
Dr. Eric Triplett
Dr. Elaine Turner
Dr. Brian Harfe
UF College Sponsors
College of Agriculture and Life Sciences
College of Liberal Arts and Sciences
References
Carpenter, S. R. (2008). Phosphorus control is critical to mitigating eutrophication. Proceedings of the National Academy of Sciences, 105(32), 11039-11040. doi:10.1073/pnas.0806112105
Dr. David Norton
Dr. Eric Triplett
Dr. Elaine Turner
Dr. Brian Harfe
UF College Sponsors
College of Agriculture and Life Sciences
College of Liberal Arts and Sciences
References
Carpenter, S. R. (2008). Phosphorus control is critical to mitigating eutrophication. Proceedings of the National Academy of Sciences, 105(32), 11039-11040. doi:10.1073/pnas.0806112105
Dodds, W. K., Bouska, W. W., Eitzmann, J. L., Pilger, T. J., Pitts, K. L., Riley, A. J., . . . Thornbrugh, D. J. (2009). Eutrophication of U.S. Freshwaters: Analysis of Potential Economic Damages. Environmental Science & Technology, 43(1), 12-19. doi:10.1021/es801217q
Williams, Sarah. "DNA Tape Recorder Stores A Cell's Memories". Science | AAAS, 2019. https://www.sciencemag.org/news/2014/11/dna-tape-recorder-stores-cells-memories