Team:UFlorida/Description
Project Description
Eutrophication
Eutrophication occurs when a body of water becomes overly enriched with nutrients, predominantly due to human industrial activity. This in turn causes excess algae growth that diminishes oxygen supply to other organisms [1]. The algal decomposition process and shading of marine plants also contribute to depletion of oxygen levels [6].
Harmful Algal Blooms
Harmful algal blooms (HABs) have had an extensive history of ravaging Florida’s water bodies. Red tide algal blooms and other HABs often lead to massacre scenes, leaving hundreds of deceased fish blanketing beaches and riverbanks. There is sufficient evidence supporting that fertilizer runoff from industrial agriculture sites is a major cause of the HABs responsible for red tide, with a major ingredient in fertilizers being phosphorous [1]. The resulting wave of death sweeping through waterways threatens the biodiversity of the ecosystems within them. The algae responsible for red tide, Karenia brevis, also emits neurotoxins after crashing onto the surf. This adds a layer of public health concern in regard to HABs, as these toxins can cause respiratory complications in humans and other land animals [4].
Source: Fleming, L. E., Kirkpatrick, B., Backer, L. C., Walsh, C. J., Nierenberg, K., Clark, J., Reich, A., Hollenbeck, J., Benson, J., Cheng, Y. S., Naar, J., Pierce, R., Bourdelais, A. J., Abraham, W. M., Kirkpatrick, G., Zaias, J., Wanner, A., Mendes, E., Shalat, S., Hoagland, P., … Baden, D. G. (2011). Review of Florida Red Tide and Human Health Effects. Harmful algae, 10(2), 224–233. https://doi.org/10.1016/j.hal.2010.08.006
The detrimental effects of HABs don’t stop here. They also devastate Florida’s economy by disrupting the tourism and fishing industries—some of the state’s most lucrative sources of income. Costs of eutrophication damages are estimated to be approximately 2.2 million dollars yearly [2]. Researchers have long established that the increased presence of phosphorus in water, as discussed by Dr. Stephen Carpenter through his research with the University of Wisconsin, is a major contributing factor to eutrophication. With more environmental and economic damage occurring with each HAB, Team Florida has found it imperative to address them at the source.
Source: Schwab, H., & Treadway, T. (2016, July 04). How toxic green slime caused a state of emergency in Florida. Retrieved October 22, 2020, from https://www.usatoday.com/story/news/nation-now/2016/07/04/how-toxic-green-slime-caused-state-emergency-florida/86679518/
How Can Team Florida Help?
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.
What is the PhoB-PhoR System?
The PhoB-PhoR genes are part of a system called the Pho regulon. This system serves to transport inorganic phosphate(pi) into Escherichia coli when these organisms lack this resource. This regulatory network has a histidine kinase sensor (PhoR) and a response regulator protein (PhoB). When E.coli cells are low in phosphate, these organisms activate gene expression of genes like PhoR which serve as a sensor protein that when stimulated by the lack of phosphate, catalyzes the activation of response regulator PhoB. As a positive response regulator, PhoB activates the gene expression of itself alongside PhoR and other genes essential for the intake of (Pi) into E.coli [7]. PhoB then goes on to directly active promoter PhoA.
Source: Uluseker, et. all. “A Dynamic Model of the Phosphate Response System with Synthetic Promoters in Escherichia coli.” Mit Press Journals, 2017. https://www.mitpressjournals.org/doi/pdf/10.1162/isal_a_069
Our Genetic Circuit
Team Florida has taken this system and added a genetic inverter to enable the PhoB-PhoR system to respond to high phosphorus levels. The genetic inverter consists of Tetr and pTet which are placed after promoter PhoA, and before SCRIBE, as seen in figure A.
This system will work as such: in low phosphate conditions, pPhoA is activated, leading to the transcription of the Tetr repressor gene. The Tetr repressor gene is then translated into the Tetr protein, which binds and represses the pTet promoter; therefore, SCRIBE is not transcribed.
But because pTet is constitutive, in high phosphate conditions when the PhoBR is turned off in E. Coli, the pTet promoter remains active due to the absence of Tetr, and the scribe system is under control of the pTet promoter. Therefore, SCRIBE is transcribed and a mutation results in the bacterial chromosome leading to the quantification of phosphorus in a given environment.
Figure a: Genetic circuit containing PhoBR, genetic inverter, and SCRIBE
Figure b: Genetic inverter consisting of Tetr and pTet
What is SCRIBE and How Does it Work?
SCRIBE consists of the following: Inducible promoter, msr, msd(rpoB), RBS, Reverse Transcriptase, RBS, Beta Recombinase Protein, and Terminator. SCRIBE is induced by environmental chemicals to make permanent mutations to the bacterial chromosome. This system gives scientists the ability to couple a chosen stimulus with a desired mutation to quantify that stimulus. In our case, the stimulus is phosphorous. 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 [8].
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 is SCRIBE Better Than Other Quantification Methods?
Great question. 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, so if there was a short influx of phosphorus, the protein expression would halt.
References
[1] 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
[2] 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
[3] Eutrophication. (n.d.). Retrieved October 22, 2020, from https://projectbaseline.org/eutrophication/
[4] Fleming, L. E., Kirkpatrick, B., Backer, L. C., Walsh, C. J., Nierenberg, K., Clark, J., Reich, A., Hollenbeck, J., Benson, J., Cheng, Y. S., Naar, J., Pierce, R., Bourdelais, A. J., Abraham, W. M., Kirkpatrick, G., Zaias, J., Wanner, A., Mendes, E., Shalat, S., Hoagland, P., … Baden, D. G. (2011). Review of Florida Red Tide and Human Health Effects. Harmful algae, 10(2), 224–233. https://doi.org/10.1016/j.hal.2010.08.006
[5] Schwab, H., & Treadway, T. (2016, July 04). How toxic green slime caused a state of emergency in Florida. Retrieved October 22, 2020, from https://www.usatoday.com/story/news/nation-now/2016/07/04/how-toxic-green-slime-caused-state-emergency-florida/86679518/
[6] The Effects: Dead Zones and Harmful Algal Blooms. (2017, March 10). Retrieved October 22, 2020, from https://www.epa.gov/nutrientpollution/effects-dead-zones-and-harmful-algal-blooms
[7] Uluseker, et. all. “A Dynamic Model of the Phosphate Response System with Synthetic Promoters in Escherichia coli.” Mit Press Journals, 2017. https://www.mitpressjournals.org/doi/pdf/10.1162/isal_a_069
[8] 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.