Team:Alma/Poster

Poster: Alma

Presenting: Poisoned River

Abstract
Our team's two-year project, Poisoned River, will focus on creating a DDT-detecting biosensor to aid researchers and cut down on costs. Several species of animals have estrogen receptors known to bind DDT, a xenoestrogen. Linking this binding process to a reporting gene, such as RFP, within a microbe will allow for the detection of organochlorines, which can enhance broad spectrum screening of these contaminated areas both locally and globally. Ultimately, this biosensor has the potential to save thousands of dollars in the pollution cleanup effort as well as provide a basis for the development of future synthetic biology tools for the bioremediation of DDT.

Contributors
The main poster contributors were Abbey Killian (author), Kaleb Ramon (author), and Kelsey Taylor (coding, formatting, proofreading, graphics).
Additional support was from Connor Arens (proofreading), Gary Carter (coding aide), Marleigh Matthews (providing previously-designed graphics), and Dr. Devin Camenares (proofreading).

Background

In the 1980s, the Velsicol Chemical Plant contaminated local waterways and properties in St. Louis, Michigan with DDT and its derivatives. The contamination was so severe, the Environmental Protection Agency (EPA) stepped in to help with what is now the largest superfund site found in Michigan. Further into the investigation, it was discovered that river sediment next to the site was 4% DDT/DDT derivatives. The pollution problem was far from solved and getting worse. In 1997, the Pine River Superfund Citizens Task force was formed as a local advisory group to the EPA. They pushed the EPA for money to clean up the site. The EPA eventually fulfilled the request and a filtration process began that still occurs today.



The goal of Poisoned River is to address the environmental havoc that has affected our community for half a century. With our two-year project, we hope to have our biosensor finished by next year’s competition and ready for active use by the Environmental Protection Agency (EPA) and other researchers that take samples from the Pine River and surrounding areas. Future projects may go beyond this projects' biosensor through researching a degradation pathway to diminish the levels of DDT and its derivatives. If this biosensor/degration pathway combination is successful, we hope to then adjust our biosensor to detect and degrade other chemicals beyond DDT.

Inspiration: Local

Alma iGEM is based in Alma College in Gratiot County, MI. Although the contamination originated at the Velsicol Chamical Plant in St. Louis in Isabella County, the pollutants stretch across county lines and have impacted local lives for far too long. We wish to relieve not only the stress on our local citizens of not being able to use these waterways, but also the environment. The strain that DDT has had on the bird populations, egg production, and the unknown adverse effects that it has on humans, has gone on too long. Upon understanding this problem and starting a communication line with the EPA, we learned more about the financial aspects of what Velsicol Chemical Plant has burdened us with. Our biosensor will not only provide environmental relief, but greatly reduce the costs of cleanup efforts by an estimated amount of over $100,000.

Problem: DDT

DDT is an environmental pollutant that was heavily used as a pesticide in the United States until it was banned from agricultural use in 1972. However, DDT is still used worldwide in developing nations as it is particularly effective at reducing rates of malaria, despite its negative effect on the environment. DDT, and structurally similar organochlorines, are known as endocrine disrupting chemicals (EDCs) that act to disrupt hormonal balance in wildlife. They are additionally characterized as xenoestrogens and potential human carcinogens. This chemical has also been noted to disrupt the reproductive cycle of avian species, as birds exposed to the chemical show a decreased ability to produce the calcium carbonate necessary for a hard eggshell. Therefore, avian populations in the local St. Louis area have been steadily declining since the closure of the Velsicol Chemical Plant. The rising need to aid in the remediation of DDT and other organochlorines in both local and global areas is why we believe constructing a biosensor will be integral to the future of the environment.

Idea: Biosensor

While in the brainstorming phase, we knew we wanted to deal with environmental pollutants, especially those found in the Pine River area. Our initial idea was actually the design of a DDT degradation pathway in order to aid directly in the remediation efforts. We decided to run this idea by Dr. Harwood, an Assistant Professor at Alma College, to get some feedback. She was able to provide us with insight as to what direction to go in. She advised us that the Pine River’s main problem is actually the contamination of the derivatives of DDT and not DDT itself; therefore, a degradation pathway of DDT would not be the most efficient route to take in terms of remediation. She instead suggested that we come up with a screening protocol to administer risk assessment to aid in clean-up efforts. She indicated this would be helpful to all the people who are researching along the Pine River, and will serve as a way to focus efforts and resources on the places that need it most. We additionally held a meeting with the three EPA superfund site coordinators he showed great intrigue and enthusiasm regarding the prospect of a biosensor, noting it would be of great use to the remediation of the superfund site.

Engineering

In order to address the organochlorine pollution that is seen at the St. Louis superfund site, we elected to undertake the creation of a biosensor that will be sensitive to organochlorines like DDT. Our genetic circuit consists of the usage of three different BioBricks assembled in series that will elicit a physical change when in the presence of DDT. The genetic circuit consists of the BioBricks I13521, K123002, and K123003, assembled in that order. I13521 acts as our reporting gene, as it is a BioBrick that is under control of a TeT repressible promoter and codes for RFP. K123002 acts as our inverter and is crucial to the design of our genetic circuit. This BioBrick is under the control of the constitutive LacI promoter and codes for the production of TetR. Therefore, this piece acts to constitutively repress the production of RFP; it also contains a sequence of DNA known as the estrogen response element, or ERE, which is crucial to its inverting power in this circuit. When the intracellular estrogen receptor is bound by estrogen -- or an estrogen analog like DDT -- this estrogen receptor complex can bind the ERE, which inhibits the transcription and translation of TetR. In turn, the I13521 part is no longer repressed and the production of RFP occurs, indicating a sample contains DDT. Finally, the K123003 codes for the intracellular human estrogen receptor alpha. Ultimately, our microbial biosensor should elicit a red fluorescent visual change when exposed to a sample containing DDT or other structurally similar organochlorines, and this fluorescence can be measured using a fluorescent plate reader in an effort to approximate the concentration of organochlorines within a tested sample. The image below shows our constructed genetic circuit.

Scot Science Podcast

In an attempt to share and spread science within our local community and those abroad, the Scot Science Podcast was created. It is recorded in a manner that would allow it to be consumable by anyone, and frequently aims to make complex subjects and topics more easily understandable for the audience. This allows a broader audience of educational backgrounds and interests to listen to the podcast. There have been interviews from multiple guest speakers ranging from Alma College professors to other iGEM teams, allowing for a greater range of influence, opinions, and expertise to be heard and distributed through the podcast.

Human Practices

To help generate ideas, we invited elementary students to our campus to ask them how they think that synthetic biology could improve their community. A few that were seen included: “stop pollution in the environment,” and “make a bacteria that eats away pollution without hurting the environment.” Although these are just a couple of examples, the wide majority of the responses were related to pollution, water, and animals in the environment.


After inviting these students to our college, we decided to visit the local Alma High School to practice different lab techniques and have a discussion during wait times about iGEM and synthetic biology. Here, we did a modern transformation lab that was able to help students review the well-known experiment by Avery and Macleod, which lined up perfectly with the class’s curriculum. Within this, students were able to learn how to perform DNA transformation

After the lab was performed, one of our students was able to sit with the high school students and talk about involvement in iGEM and what synthetic biology could do for our community. During this time, our team was tossing around the idea of the Pine River and we received positive feedback from the students who grew up in the community, saying that they were excited that we may go with this topic.


Right before the nationwide mandatory COVID-19 quarantine was enforced, we were able to visit a science fair at Greenspire, a STEM-focused middle school. Here, we set up a booth to explain the mini-lab that we had set up, which involved the use of E.coli transformed with fluorescent proteins and chromoproteins to form a type of “bacterial art” by streaking the bacteria on agar plates. This allowed us to form an ongoing connection with these students, as they provided contact information so that we can send them images of their plates after incubation. We were able to teach these students not only about the E.coli strain we were using and how we were able to have multiple colors of the bacteria, but also about safety techniques when handling bacteria.

Future Directions

The initial intention of our constructed biosensor was to be able to use it as a broad spectrum screening tool in order to aid the remediation efforts at the St. Louis superfund site. However, the potential for much further design implementations has been theorized. Firstly, the use of an additional reporting gene, such as GFP, can be linked to another inverter in order to offer a semi-quantitative analysis of samples. The EPA has guidelines that show concentrations of DDT at 40 ppm or higher call for emergency response. Therefore, the use of both of these reporting genes in a single genetic circuit with multiple EREs would allow for the production of one fluorescent protein at certain concentrations of DDT (below 40 ppm) and the production of both fluorescent proteins at concentrations that exceed this level. This would rely heavily on the optimization of our constructed math model for our circuit as we need fairly precise control of protein expression in order for this circuit to work. We have additionally theorized using multiple species-specific estrogen receptors within a single circuit linked to individual reporting genes in order to indicate the presence of differing xenoestrogens within a tested sample. This would allow the expansion of our circuit to cover for an increased amount of endocrine disrupting chemicals aside from just DDT and its derivatives, meaning our biosensor could be applied much more broadly to global remediation efforts outside of the St. Louis superfund site.

Photo was taken pre-COVID-19.

Results

Prior to assembling the three pieces, further characterization of each individual piece was undertaken. I13521 was shown to be properly controlled by its TeT repressible promoter when transformed into two separate strains of E. coli: one that constitutively expresses TetR and one that does not. These two strains showed that in the presence of TetR, I13521 is shut off, but when not in the presence of TetR, RFP is produced as can be seen below.

Additionally, SDS PAGE was conducted on both I13521 and K123002 in order to characterize protein expression by both parts independently. I13521 showed an expression of RFP; however, K123002 did not have any visible band corresponding to TetR. Therefore, further investigation into this piece must be performed in order to address this. We additionally attempted to characterize K123003, but we were unable to successfully transform it into NEB stable strain E. coli. However, colony growth was seen in our positive controls, so the possibility of cytotoxicity is being investigated. Our first steps in this process include codon harmonizing the sequence of the human estrogen receptor alpha in order to aid in the translation of the protein. Finally, we were able to successfully assemble I13521 and K123002 together through Gibson assembly to create a new BioBrick: K3445001. This piece was also characterized by SDS PAGE analysis. At baseline, we would expect our assembled BioBrick to be repressing the production of RFP. However, contrary to this idea, we see the alternative. Colonies containing our newly assembled BioBrick were in fact red in color and did demonstrate a SDS PAGE band corresponding to RFP. The uninhibited production of RFP could be explained by our SDS PAGE finding for K123002, which showed no TetR present. Therefore, it is possible our circuit is either expressing TetR at levels too low to repress the TeT repressible promoter or the degradation tag on the expressed TetR is too powerful and causing the degradation of TetR before it can repress the production of RFP. Further investigation is warranted. The SDS PAGE analysis can be seen below, with an arrow indicating a protein size of approximately 25 kDa: the approximate size of both RFP and TetR.

References

Kenneth, Davis S. “The Deadly Dust: The Unhappy History Of DDT.” AMERICAN HERITAGE, American Heritage Publishing Co., Feb. 1971, www.americanheritage.com/deadly-dust-unhappy-history-ddt.

McMacked, David. History by Decades, City of St. Louis, 2003, \www.stlouismi.com/1/stlouis/history_by_decades.asp.
The article is a compilation of excerpts from the book "St. Louis at 150-The story of the Middle of the Mitten" by Daniel McMacken (2003).

"Improved Part. Making a better estrogen sensor,"Carnegie Mellon iGEM Team, 2015, https://2015.igem.org/Team:Carnegie_Mellon/improvedpart.

"Sensor That Reports Endocrine Activating Molecules,"Carnegie Mellon iGEM Team, 2014, https://2014.igem.org/Team:Carnegie_Mellon/Our_Sensor.

Felton, Rachel G., et al. “Identification of California Condor (Gymnogyps Californianus) Estrogen Receptor Variants and Their Activation by Xenoestrogens.” General and Comparative Endocrinology, Academic Press, 1 Apr. 2020, www.sciencedirect.com/science/article/pii/S0016648019305581?via=ihub.

Liang, Rubing, et al. “Construction of a Bacterial Assay for Estrogen Detection Based on an Estrogen-Sensitive Intein.” Applied and Environmental Microbiology, American Society for Microbiology, 1 Apr. 2011, aem.asm.org/content/77/7/2488.

Matthews, Jason, et al. “Differential Estrogen Receptor Binding of Estrogenic Substances: a Species Comparison.” The Journal of Steroid Biochemistry and Molecular Biology, Pergamon, 8 Jan. 2001, www.sciencedirect.com/science/article/pii/S0960076000001266.

Vincent, O'Brien, et al. “Designing a Biosensor to Detect Endocrine-Disrupting Compounds.” St. Mary's College of California, 2012.

Sponsors
Revive & Restore
Alma College Biochemistry Department
Alma College New Media Studies Department
Alma College Student Congress
New England Biolabs
Integrated DNA Technologies
MathWorks
Geneious
SnapGene