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<h2>Research plan summary</h2>
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<p>PFAS (per and poly fluorinated alkyl substances) are an increasingly prevalent threat to humans and animals. PFAS are industrial products that are extremely persistent, resilient to degradation, and widespread in the environment, especially in the United States and our home state of Kentucky. PFAS has been responsible for a variety of human diseases, such as cancers, birth defects, autoimmune responses, and many more characterized and potentially uncharacterized impacts. PFAS testing is also very inaccessible, requiring mass spectroscopy machines that not many places have. Our project combats this problem by creating a gene circuit that causes transformed bacteria to fluoresce in the presence of PFAS, providing an accessible in situ test for PFAS. Additionally, our team also sought to gain a deeper understanding of the methods available to detect PFAS through literature searches of the molecular mechanisms of PFAS toxicity, reverse screening of proteins affected by PFAS, kinetic modeling of our gene circuit, and molecular dynamics modeling of PFAS interactions with potential signal proteins. Our team also had to overcome challenges related to the size and complexity of our gene circuit, problems associated with being a first year team, and delays in lab proceedings. Although we were unable to finish lab testing, we successfully completed a kinetic simulation of our gene circuit which can be viewed or modified by anyone else freely. We also gained a deeper understanding of the molecular mechanisms of PFAS through computational simulations and literature searches, the results of which are published on this wiki. </p>
<pstyle="margin-top: 3vh;">Dubbed per and polyfluorinated alkyl substances, PFAS are an issue in our environment. These chemicals were first discovered during experimentation with refrigerator gases and soon became commonplace in every citizen’s life. DuPont chemical company became a behemoth with the help of PFOA purchases from 3M to make their revolutionary nonstick spray, Teflon. Our group began by talking to prospective PIs at our local university (University of Louisville). We saw that one of them had researched PFAS detection in wastewater in association with graduate students. This research inspired us to pursue a PFAS-related topic, and we wanted to address the root of the issue in the United States: the ubiquity of the chemical across all spheres of life. From firefighters using aerosol cans to the typical, household, nonstick cooking spray, PFAS is everywhere. The issue with this chemical lies in its fundamental structure. PFAS chemicals contain carbon-fluorine chains, which are strong bonds. These bonds hold the chemical together despite environmental intervention, causing the chemical to be resistant to degradation. When ingested through the residue from the nonstick cooking spray on food, for example, the chemical bioaccumulates, which is increasingly problematic. The bioaccumulation of this chemical can lead to the effects listed above in the abstract and is prevalent at a community level in West Virginia. In this state, PFAS waste was dumped into local waterways, leading to congenital disabilities in children. With the DuPont factory being one of the largest employers in the area, many employees were also affected by chronic diseases like diabetes. Our group was shocked to see disparate access barriers in PFAS testing kits. After reading the story the movie “Dark Waters” was based on, we were appalled by the loss of livestock for farmers within seconds due to polluted waterways. Although a first-year team, we set out on an ambitious goal to model a mass detection system for PFAS using genetic engineering, which would create equitable access to clean water. Even though we live in Louisville and are fortunate enough to have a company as great as the Louisville Water Company filter our water, we wanted to make an impact across all communities that might not be as fortunate as ours and ensure that clean water was available to those in future generations to come.</p>
<pstyle="margin-top: 3vh;">The USAFA 2019 team used a PFAS-sensitive promoter to upregulate the transcription of mRFP mRNA. Our PFAS-detecting gene construct uses the AHL quorum sensing molecule as a way to amplify the signal produced by the PFAS-sensitive prmA-promoter. AHL first needs to bind with a protein called LuxR before it can influence transcription of the pLux promoter. This amplification scheme was attempted by the Stockholm 2020 team and while they tested individual components of the system, they never tested it all together. Our gene circuit will contain LuxR under constitutive transcription, LuxI (an AHL synthase) under the prmA promoter, and GFP under pLux promoter. This way, PFAS should increase the level of AHL in the cell which then binds to the abundant LuxR which finally induces large scale transcription of GFP. Each LuxI mRNA molecule transcribed as a result of PFAS detection will eventually have a larger impact on cell fluorescence than if 1 molecule of GFP mRNA was produced instead of LuxI mRNA.</p>
<p>We also created an alternative circuit with the pLac promoter instead of prmA promoter to act as a way to check if the prmA promoter was the failure point in the circuit, however we did not have enough time to test it.</p>
<p>Since the mechanism of prmA-promoter activation in response to PFAS has not been explicitly described, we conducted a literature search to propose possible mechanisms. We also conducted reverse screening of proteins affected by PFAS since PFAS toxicity has not been fully characterized yet and to find potential receptors that could be sensitive to PFAS for future works.</p>
<pstyle="margin-top: 3vh;">We created a model in Virtual Cell (VCell) to simulate the kinetics of our gene circuit. With this model we could predict the detection threshold for PFAS as well as provide a tool to future teams that wish to work with PFAS detection or the AHL-LuxR transcription control network.</p>
<p>We conducted literature searches for the rate constants used in each step of the model and estimated uncharacterized reaction constants. We also performed sensitivity analysis for certain estimated reaction rates, though sensitivity analysis for all rate constants could be conducted with VCell. Our VCell model is freely available to anyone. </p>
<pstyle="margin-top: 3vh;">Our initial objective with molecular dynamics simulations was to investigate whether PFAS (Per- and Polyfluoroalkyl Substances) could induce similar conformational changes in receptor proteins as their natural ligands. Molecular dynamics simulations involve the detailed modeling of molecular interactions and motions at the atomic level. In our case, these simulations allowed us to observe how PFAS molecules interact with and influence the structural dynamics of receptor proteins. We chose to simulate a docked complex produced by a server instead of just using the docked complex straight out of the server because of many reasons. OpenMM provides us with a high degree of customization and control over the simulation parameters. This level of flexibility allows us to fine-tune the simulation conditions to closely mimic real-world scenarios and experimental conditions. By adjusting factors such as temperature, pressure, and force field parameters, We really want the simulation to run under physiological conditions, STP. Simulating the docked complex with OpenMM enables us to gain dynamic insights into the behavior of the complex over time. This dynamic perspective is crucial for understanding how the PFAS and receptor proteins interact, evolve, and adapt within the binding site. It provides a more comprehensive view of the binding process compared to a static pre-generated complex. By running our own simulations, we can validate and verify the accuracy and reliability of the binding configuration. This process helps ensure that the docked complex is energetically stable and that the binding interactions are consistent with our expectations and scientific hypotheses. OpenMM allows us to explore a range of simulation scenarios. We can conduct multiple simulations with variations in parameters or starting conditions to assess the robustness and reliability of the predicted binding, and to consider different binding pathways or binding site conformations. Running our simulations provides a means of quality assurance. It allows us to independently verify the results, ensuring that the complex generated by the server is a valid starting point for further analysis. This exploration aimed to shed light on whether PFAS would bind as expected or introduce potential disruptions, potentially unveiling proteins capable of effectively binding to PFAS, thus serving as a fundamental mechanism for detection. To address this inquiry, we chose to simulate the binding of PFOA to the LuxR protein, primarily because of its resemblance to the nonpolar nature of LuxR's natural ligand, AHL. If PFOA could successfully bind to LuxR, it would establish a direct means of detecting PFAS within cells, significantly streamlining our research process and minimizing the need for extensive modifications. However, it's essential to acknowledge that our current findings remain speculative, and ongoing efforts are dedicated to refining our molecular dynamics simulations, ensuring increased accuracy, and generating more comprehensive insights and data.</p>
<p>Describe how and why you chose your iGEM project.<p>
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<p>Please see the <ahref="https://competition.igem.org/judging/medals">2023 Medals Page</a> for more information.</p>
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<h2>Research plan summary</h2>
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<p>
Our team will tackle the growing problem of PFAS (poly and per-fluoroalkyl substances) contamination/pollution. PFAS, due to its chemical inertness, are extremely hard to decompose and has been named “forever chemicals”. These substances have become more and more widespread to the point where 97% of Americans have detectable amounts of it in their blood (NHANES 2015). The health risk of PFAS is not fully understood but has been implicated in liver diseases, cancers, increased cholesterol levels, and other complications. Our team attempts to help combat this problem by engineering bacteria to produce an observable signal when they encounter PFAS. PFAS are usually detected with liquid or gas chromatography and mass spectrometry, which is time consuming, expensive, and inaccessible to the public. PFAS-sensitive bacteria have the potential for extremely high-throughput testing for PFAS. For example, samples of water can be parallelly tested in well plates by incubating each sample with bacteria and looking for the signal. Alternatively, a small box containing the bacteria and a rudimentary fluorescence detector could be used by farmers or mobile water testers to quickly test samples. Such devices could even be installed in waterways downstream of chemical plants and passively monitor PFAS presence. We hope to be able to make a system based on this genetic circuit that can make PFAS detection more accessible and faster so that PFAS contamination can be quickly found and contained before more damage is caused.
We mainly drew on the research conducted by 2 former iGEM teams: Stockholm 2020 and the US Air Force Academy (USAFA) teams from 2019-2021. We and the USAFA 2019 team also drew on a paper (Weathers et al., 2015) that described how the promoter for the prmA gene found in Rhodococcus jostii. RHA1 upregulated transcription in response to PFOA (Perfluorooctanoic acid, a type of PFAS). With this knowledge, the USAFA 2019 team engineered a plasmid that put the mCherry reporter protein gene under the control of the prmA promoter and transformed it into bacteria. They then exposed this bacteria to PFOA and measured activation of the promoter. Although no fluorescence could be detected, they found that levels for mRNA coding for mCherry was roughly twice as high in the bacteria exposed to 1 micromolar PFOA than the control sample in plan LB broth, thus showing the prmA promoter could be used to detect PFOA presence.
The Stockholm team attempted to amplify the signal from the prmA promoter by using quorum sensing autoinducer molecules, specifically 3OC6HSL. Essentially, they placed the autoinducer synthase, LuxI, under the control of the prmA promoter. Then, they placed a reporter gene that would produce electricity under the control of a promoter sensitive to 3OC6HSL combined with a regulator protein LuxR produced in a different segment. While they did not test the full genetic circuit, they found that 3OC6HSL produced by other cells was able to trigger the 3OC6HSL sensitive promoter (named Plux). However, they were unable to detect any upregulation of genes controlled by the prmA promoter when exposing transformed bacteria with PFOA by using western blot. Since the Stockholm team already characterized the effectiveness of the Plux promoter, our team decided to use a similar approach to the Stockholm team to amplify the signal of the prmA promoter. Instead of producing electricity, we will simply express GFP, however we will use the same Plux promoter and the same quorum sensing autoinducer 3OC6HSL to amplify the signal. It is worth noting that 3OC6HSL diffuses through bacterial cell membranes so it may be able to have a very pronounced effect in increasing PFAS sensitivity.
Where Pconst is a constitutive promoter, RBS are ribosomal binding site sequences, and (stop) is a double terminator sequence.
Ideally, exposure to PFAS will cause the LuxI gene to be expressed which then makes 3OC6HSL. These autoinducers will bind to the constitutively expressed LuxR inducer protein and that complex will bind with the pLux promoter which will upregulate transcription of GFP. We hope this cascade of signals, from PFAS to 3OC6HSL, will increase the amount of GFP expressed in response to PFAS exposure.
We plan to order these sequences from DNA synthesizing companies such as Integrated DNA Technologies or Twist Biosciences which may come at a considerable cost. The DNA will be inserted into plasmids and then transformed into E. coli via electroporation.
We also designed a construct to test the amplification effectiveness of the circuit by replacing the prmA promoter with the lac promoter. This way, we can determine if a faulty prmA promoter sequence causes our bacteria to be insensitive.
Below are the iGEM parts and sequences for each component of our genetic circuit. <br>
Pconst(BBa_J23100)<br>
RBS(BBa_B0034)<br>
LuxI(BBa_C0061)<br>
Pprma (BBa_K2911000)<br>
DT(BBa_B0015)<br>
Plux (BBa_R0062)<br>
GFP(BBa_E0040)<br>
LuxR(BBa_C0062)<br>
pLac(BBa_R0010)<br>
In order to determine the sensitivity of our designed circuit we plan to conduct two lines of experimentation, one that uses the first gene construct and the second that uses the second gene construct with the lac promoter instead of the prmA promoter.
The first line of experiments consists of incubating PFAS-sensitive transformed bacteria in different concentrations of PFOA, namely 0, 0.5, 1, 50, and 100 micromolar. Cell samples should be drawn at 8 hour intervals and subjected to both fluorescence reading and qRT-PCR for mRNA coding for GFP.
The second line of experiments uses IPTG-sensitive bacteria transformed with the second genetic construct. These bacteria are incubated in 0, 0.1,0.5, 1, and 50 mM IPTG. samples are similarly drawn at 8 hour intervals and subjected to fluorescence detection and qRT-PCR for mRNA coding for GFP. The purpose of these experiments is to test the sensitivity of our system to a well-characterized compound and compare it to the minimum detection limit of IPTG by E. coli published in the literature.
<p>We encourage you to put up a lot of information and content on your wiki, but we also encourage you to include summaries as much as possible. If you think of the sections in your project description as the sections in a publication, you should try to be concise, accurate, and unambiguous in your achievements. Your Project Description should include more information than your project abstract.</p>
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<h2>References</h2>
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<p>iGEM teams are encouraged to record references you use during the course of your research. They should be posted somewhere on your wiki so that judges and other visitors can see how you thought about your project and what works inspired you.</p>
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Our team will tackle the growing problem of PFAS (poly and per-fluoroalkyl substances) contamination/pollution. PFAS, due to its chemical inertness, are extremely hard to decompose and has been named “forever chemicals”. These substances have become more and more widespread to the point where 97% of Americans have detectable amounts of it in their blood (NHANES 2015). The health risk of PFAS is not fully understood but has been implicated in liver diseases, cancers, increased cholesterol levels, and other complications. Our team attempts to help combat this problem by engineering bacteria to produce an observable signal when they encounter PFAS. PFAS are usually detected with liquid or gas chromatography and mass spectrometry, which is time consuming, expensive, and inaccessible to the public. PFAS-sensitive bacteria have the potential for extremely high-throughput testing for PFAS. For example, samples of water can be parallelly tested in well plates by incubating each sample with bacteria and looking for the signal. Alternatively, a small box containing the bacteria and a rudimentary fluorescence detector could be used by farmers or mobile water testers to quickly test samples. Such devices could even be installed in waterways downstream of chemical plants and passively monitor PFAS presence. We hope to be able to make a system based on this genetic circuit that can make PFAS detection more accessible and faster so that PFAS contamination can be quickly found and contained before more damage is caused.
We mainly drew on the research conducted by 2 former iGEM teams: Stockholm 2020 and the US Air Force Academy (USAFA) teams from 2019-2021. We and the USAFA 2019 team also drew on a paper (Weathers et al., 2015) that described how the promoter for the prmA gene found in Rhodococcus jostii. RHA1 upregulated transcription in response to PFOA (Perfluorooctanoic acid, a type of PFAS). With this knowledge, the USAFA 2019 team engineered a plasmid that put the mCherry reporter protein gene under the control of the prmA promoter and transformed it into bacteria. They then exposed this bacteria to PFOA and measured activation of the promoter. Although no fluorescence could be detected, they found that levels for mRNA coding for mCherry was roughly twice as high in the bacteria exposed to 1 micromolar PFOA than the control sample in plan LB broth, thus showing the prmA promoter could be used to detect PFOA presence.
The Stockholm team attempted to amplify the signal from the prmA promoter by using quorum sensing autoinducer molecules, specifically 3OC6HSL. Essentially, they placed the autoinducer synthase, LuxI, under the control of the prmA promoter. Then, they placed a reporter gene that would produce electricity under the control of a promoter sensitive to 3OC6HSL combined with a regulator protein LuxR produced in a different segment. While they did not test the full genetic circuit, they found that 3OC6HSL produced by other cells was able to trigger the 3OC6HSL sensitive promoter (named Plux). However, they were unable to detect any upregulation of genes controlled by the prmA promoter when exposing transformed bacteria with PFOA by using western blot. Since the Stockholm team already characterized the effectiveness of the Plux promoter, our team decided to use a similar approach to the Stockholm team to amplify the signal of the prmA promoter. Instead of producing electricity, we will simply express GFP, however we will use the same Plux promoter and the same quorum sensing autoinducer 3OC6HSL to amplify the signal. It is worth noting that 3OC6HSL diffuses through bacterial cell membranes so it may be able to have a very pronounced effect in increasing PFAS sensitivity.
Where Pconst is a constitutive promoter, RBS are ribosomal binding site sequences, and (stop) is a double terminator sequence.
Ideally, exposure to PFAS will cause the LuxI gene to be expressed which then makes 3OC6HSL. These autoinducers will bind to the constitutively expressed LuxR inducer protein and that complex will bind with the pLux promoter which will upregulate transcription of GFP. We hope this cascade of signals, from PFAS to 3OC6HSL, will increase the amount of GFP expressed in response to PFAS exposure.
We plan to order these sequences from DNA synthesizing companies such as Integrated DNA Technologies or Twist Biosciences which may come at a considerable cost. The DNA will be inserted into plasmids and then transformed into E. coli via electroporation.
We also designed a construct to test the amplification effectiveness of the circuit by replacing the prmA promoter with the lac promoter. This way, we can determine if a faulty prmA promoter sequence causes our bacteria to be insensitive.
Below are the iGEM parts and sequences for each component of our genetic circuit. <br>
Pconst(BBa_J23100)<br>
RBS(BBa_B0034)<br>
LuxI(BBa_C0061)<br>
Pprma (BBa_K2911000)<br>
DT(BBa_B0015)<br>
Plux (BBa_R0062)<br>
GFP(BBa_E0040)<br>
LuxR(BBa_C0062)<br>
pLac(BBa_R0010)<br>
In order to determine the sensitivity of our designed circuit we plan to conduct two lines of experimentation, one that uses the first gene construct and the second that uses the second gene construct with the lac promoter instead of the prmA promoter.
The first line of experiments consists of incubating PFAS-sensitive transformed bacteria in different concentrations of PFOA, namely 0, 0.5, 1, 50, and 100 micromolar. Cell samples should be drawn at 8 hour intervals and subjected to both fluorescence reading and qRT-PCR for mRNA coding for GFP.
The second line of experiments uses IPTG-sensitive bacteria transformed with the second genetic construct. These bacteria are incubated in 0, 0.1,0.5, 1, and 50 mM IPTG. samples are similarly drawn at 8 hour intervals and subjected to fluorescence detection and qRT-PCR for mRNA coding for GFP. The purpose of these experiments is to test the sensitivity of our system to a well-characterized compound and compare it to the minimum detection limit of IPTG by E. coli published in the literature.
