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Collaborations @@ -188,7 +189,9 @@ suggestion, our team made modifications to the Current Solutions section and also decided to include an additional page recapitulating the ways in which our project covers the weak points of other solutions.

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On September 22nd, Cornell iGEM held a Skype meeting with the members of W&M iGEM to discuss the integration of their curli fibers into our bioreactor. Since curli fibers enable biofilm development, our team sought to use them to improve the efficiency of trapping our bacteria in alginate beads. After we received the DNA parts from W&M iGEM, we tested whether the efficiency of our bioreactor was higher with or without the curli fibers. [results?] -
+

Our team also forwarded our alginate bead formation protocol to W&M iGEM so they could look for the experimental conditions that promote optimized bacterial adhesion onto the alginate beads. [results?]

@@ -239,32 +242,33 @@ engagement surveys as a way to broaden the scope of our respective projects by gaining feedback from an international perspective.

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  • Alliance for Science
  • Social Media Outreach
  • Knowledge Collaboration
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  • Harmful Algal Blooms Informational Booklet
    • @@ -281,7 +282,35 @@ DNA, so the kids were able to understand why they were adding dish soap and isopropyl alcohol to the strawberry liquid. From this point, we drew parallels between the protocols used in wet lab work to the extraction they just accomplished with everyday household materials. +

    + At the gel electrophoresis station, we discussed the purpose and process of performing gel electrophoresis + in the lab before showing them how to properly use a micropipette. We then had each kid try pipetting a + small amount of juice into an agarose gel. +

    + For the bioethics activity, we prepared three case studies that examined issues relevant and urgent to the + synthetic biology community. Our goal with this station was to open discussions to address any + misconceptions and answer any questions the students might have about synthetic biology. The students were + very receptive to the prompts and brought up key points that at times were even overlooked by the case + studies. It was also encouraging to see some of the adults who were supervising the students during the + event participate in the conversations to share their multi-generational perspectives and understandings of + how synthetic biology impacts the greater community. +

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    + We wrapped up each session with a Kahoot quiz, which is an interactive online quiz that students can + participate in using their mobile devices, to review the concepts taught at each station. The Kahoot was a + huge success since many students were already familiar with this popular teaching tool that allows students + to partake in a friendly competition to see who knows the most about the quiz topics. In our case, the + Kahoot provided an additional incentive for the students to pay close attention during the mini-lessons at + each station where members of our iGEM team explained the science behind our prepared activities. Through + the 4-H Careers Exploration event, we were able to spark conversations about different career options in the + field of synthetic biology and get the students excited about its endless possibilities.

    +

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    @@ -370,6 +399,21 @@ resources and practical guidance for students working on projects in synthetic biology.

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    Harmful Algal Blooms Informational Booklet
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    + Cornell iGEM created an informational booklet on the subject of harmful algal blooms to teach community + members about the background, prevalence, and existing treatment methods for the issue. We also included a + section that touched on the details of our project and how reHAB provides a more efficient and effective + method of detection and remediation of microcystins. The PDF of our booklet can be found under the + corresponding section of our Collaborations + page. +

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    Mlr Gene Cassette (BBa_k2960001-11)
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    + A cassette of proteins endogenous to Sphingopyxis sp. has been shown to sequentially break down microcystin-LR (Figure 1). The proteins have been named mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF [2]. The genes for these proteins are the basis of our composite biobricks. +

    + +
    Figure 1. MC-LR degradation pathway.


    +

    + MlrA is the first enzyme in the microcystin-degradation pathway. It is a microcystinase that degrades cyclic microcystin-LR to form a linear intermediate. To do so, mlrA hydrolyzes the peptide bond between Adda at position 5 and the amino acid residue at position 4 [2].

    + MlrB is the second enzyme in the microcystin-degradation pathway. MlrB is a serine protease that catalyzes the hydrolysis of linearized MCs via the cutting of the peptide bond between alanine at position 1 and a variable amino acid (e.g. leucine) residue at position 2. This results in the formation of a tetrapeptide that is subsequently degraded by MlrC [2].

    + MlrC is the third enzyme in the microcystin-degradation pathway. MlrC is assumed to be a metallopeptidase. It cleaves the tetrapeptide intermediate into an Adda amino acid and other smaller peptides. The exact smaller peptides that result are still unknown [2, 3, 4, 5].

    + MlrD codes for what is most likely a transmembrane transporter protein responsible for transporting the cyclic microcystin-LR toxin from the extracellular environment into the cell. However, its exact function is yet to be characterized. Although mlrD does not ultimately play a role in our project, we have added this part so that the entire mlr cassette is documented in the Registry [2].

    + MlrE has recently been identified in the genome of Sphingopyxis sp. strain C-1 and may be involved in microcystin degradation. Its exact function is still unknown. The gene for mlrE is, however, close in proximity to the gene for mlrB, so it is hypothesized that mlrE enhances the function of mlrB (or vice versa), [6].

    + MlrF has recently been identified in the genome of Sphingopyxis sp. strain C-1 and may be involved in microcystin degradation; is the expected sixth enzyme in the microcystin-degradation pathway. Its exact function is still unknown but it likely is involved with catalyzing reactions downstream to those of mlrA through C [6].

    + For the exception of mlrD, the transporter protein, we have also included the TorA twin-arginine translocation tag in each composite part. The twin-arginine translocation (Tat) pathway is responsible for the export of folded proteins across the cytoplasmic membrane of bacteria into the periplasmic space (Figure 2). The Tat pathway acts separately from the general secretory pathway, which transports proteins in an unfolded state. A specific signal peptide, which contains three domains: a positively charged N-terminal domain, a hydrophobic domain, and a C-terminal domain, is necessary to initiate protein export by the Tat pathway [1]. +

    + +
    Figure 2. Schematic of TorA twin-arginine translocation pathway altered to transport folded mlr proteins.


    +

    + We have included this tag with the goal of transporting the microcystin-degrading enzyme into the bacterial periplasmic space. This biobrick was designed for expression in bacteria contained in a bioreactor. Moving the enzymes closer to the periphery of the bacterial cell, and therefore closer to the flow of contaminated water passing through the bioreactor, increases the efficiency of the degradation.

    + As an additional note, we decided to utilize the Twin-Arginine Translocation pathway because of its ability to transport fully folded proteins. Many proteins (such as GFP), are unable to fold properly if exported unfolded into the periplasm. The periplasm is an oxidizing environment, which promotes the formation of aberrant disulfide bonds. This can cause proteins to misfold, aggregate, and become inactive. By exporting our enzymes fully-folded into the periplasmic space, we avoid this problem.

    + Moreover, inclusion of the tag negates the need to transport individual MC-LR proteins into the cell with mlrD, which may further slow the degradation process. Furthermore, we do not need to express the entire mlr casette in each cell, which would place a high metabolic strain on the cell. Instead, we can engineer the cells to produce only one of the mlr protein species. These different cell types are then encapsulated in alginate beads and packed sequentially in the bioreactor. When microcystin-contaminated water is passed through the reactor, it is first degraded by mlrA, then these byproducts digested by mlrB, and so on. + Finally, we have also included a 3X FLAG tag at the end of the sequence. This permits protein expression to be detected, quantified and/or purified by western blot, SDS-PAGE, and other methods.

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    Aptamer (BBa_k2960000)
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    + Aptamers are oligonucleotide or peptide molecules that bind to a target molecule with high affinity, specificity and selectivity. They are widely used for environmental monitoring as “chemical antibodies.” We chose to use a DNA aptamer because it is easy to synthesize, small in size, and of excellent chemical stability.

    + Our aptamer codes for a ssDNA aptamer which binds with high specificity to microcystin-LR.

    + The aptamer was used to create a rapid colorimetric microcystin-detection sensor. Gold nanoparticles (AuNPs) were adsorbed onto the aptamer surface. Normally, in the presence of NaCl, AuNPs aggregate, shifting the solution color from red to blue. Adsorption of the AuNPs onto the aptamer, however, prevents aggregation.

    + In the presence of microcystin, the aptamer dissociates from the AuNPs and preferentially binds to the microcystin. Thus, the AuNPs will again aggregate when induced by salt. This results in a predictable shift in the absorption spectra of a solution containing microcystins [7, 8].

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    Figure 3. Binding of aptamer prevents salt-induced aggregation (and resulting color change) of AuNPs. In the presence of MC-LR, the aptamer dissociates from the AuNPs and aggregation occurs.


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    References
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    + 1. Lee, P. A., Tullman-Ercek, D., & Georgiou, G. (2006). The bacterial twin-arginine translocation pathway. Annual review of microbiology, 60, 373–395. doi:10.1146/annurev.micro.60.080805.142212
    + 2. Zhang, J., Lu, Q., Ding, Q., Yin, L., & Pu, Y. (2017). A Novel and Native Microcystin-Degrading Bacterium of Sphingopyxis sp. Isolated from Lake Taihu. International journal of environmental research and public health, 14(10), 1187. doi:10.3390/ijerph14101187
    + 3. Family M81. (n.d.). Retrieved from https://www.ebi.ac.uk/merops/cgi-bin/famsum?family=M81.
    + 4. Cerdà-Costa, N., & Gomis-Rüth, F. X. (2014). Architecture and function of metallopeptidase catalytic domains. Protein science : a publication of the Protein Society, 23(2), 123–144. doi:10.1002/pro.2400
    + 5. BLAST: Basic Local Alignment Search Tool. (n.d.). Retrieved from https://blast.ncbi.nlm.nih.gov/Blast.cgi#alnHdr_BAI47772.
    + 6. Wang, J., Wang, C., Li, J., Bai, P., Li, Q., Shen, M., … Zhao, J. (2018). Comparative Genomics of Degradative Novosphingobium Strains With Special Reference to Microcystin-Degrading Novosphingobium sp. THN1. Frontiers in Microbiology, 9. doi: 10.3389/fmicb.2018.02238
    + 7. Li, X., Cheng, R., Shi, H., Tang, B., Xiao, H., & Zhao, G. (2016). A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples. Journal of Hazardous Materials, 304, 474–480. doi: 10.1016/j.jhazmat.2015.11.016
    + 8. Aptamer-Based Biosensors to Detect Aquatic Phycotoxins and Cyanotoxins Isabel Cunha 1,*, Rita Biltes 1 , MGF Sales 2,3 and Vitor Vasconcelos 1,4 +

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  • Safety and Risk Assessment
  • Bioethics
  • Economic Analysis
    • +
  • References
    • @@ -146,14 +148,10 @@
    -
    Overview and Key Takeaways
    +
    Our Approach


    - -
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    Our Approach

    Everybody advocates for clean drinking water and pristine lakes, but not everybody would be comfortable using synthetic biology to achieve these goals. Our team examined the current legislation in place to @@ -164,16 +162,15 @@ on the frontlines combating the effects of HABs in the Finger Lake region, to ensure we have a comprehensive understanding of the situation so we can harness the full potential of our proposed system.

    -

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    Key Takeaways
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    Key Takeaways

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      +
      • Legislation on HABs have been in place for years, but recently, the interest in HABs has increased substantially. Legislative proposals are being considered to address the increase in HAB occurrences, - especially around the Cayuga Lake region.
        • + especially around the Cayuga Lake region.
      • Although current methods of prevention and treatment are sufficiently effective, they are also costly and time-consuming. New systems to detect and breakdown microcystins would improve both safety and - cost-effectiveness.
        • + cost-effectiveness.
      • HABs are dangerous to humans and are of increasing concern, mainly due to climate change that has likely contributed to more frequent appearances.
      @@ -188,20 +185,20 @@

      To take reHAB to widespread use, our project needs to abide by current legislation for HAB remediation. We first looked into the legislature on the federal level regarding water quality. The Clean Water Act of 1972 - set standards for chemical discharges from companies, but it wasn’t until 1998 that Congress recognized the - severity of harmful algal blooms and passed the Harmful Algal Bloom and Hypoxia Research and Control Act - (HABHRCA). + set standards for chemical discharges from companies [1], but it wasn’t until 1998 that Congress recognized + the severity of harmful algal blooms and passed the Harmful Algal Bloom and Hypoxia Research and Control Act + (HABHRCA)[2].

      The HABHRCA, passed in 2004 and most recently re-authorized in 2017, provides funding for the National Oceanic and Atmospheric Administration (NOAA) to continue research on the ability to detect, monitor and assess HABs. For this purpose, 13.5 million dollars was allocated to the NOAA, with this amount increasing - in small increments until 2023. + in small increments until 2023 [3].

      While these legislative acts helped us key into the current state of HAB remediation research, we found that the primary effort for HAB remediation takes place at the state and local level. For the State of New York, - Governor Andrew Cuomo introduced a four-point initiative to combat HABs in Upstate New York. While the - initiative provides $65 million to identify, prioritize and develop action plans for vulnerable lakes, the - efforts are largely focused on the prevention and identification of blooms, whereas our project is more + Governor Andrew Cuomo introduced a four-point initiative to combat HABs in Upstate New York [4]. While the + initiative provides $65 million to identify, prioritize and develop action plans for vulnerable lakes [5], + the efforts are largely focused on the prevention and identification of blooms, whereas our project is more concerned with the detection and remediation of blooms already in existence.

      Focusing on local policies here in Ithaca, our team decided to attend a public meeting of the Planning, @@ -224,6 +221,7 @@ more central focus on the issue, there is still a lack of comprehensive legislation, as many initiatives are still being discussed. By working with legislators and community leaders, our team hopes to use our project to push these initiatives forward to provide clean and safe waters for our community and the world. +

    @@ -240,22 +238,25 @@ usage, and disposal or recycling of the chemical or product. Risk assessment (RA) is advantageous in that it specifically quantifies the risk, providing a resolution through the analysis. CEA aims to combine the strengths of both assessment techniques by providing a guideline for organizing information from various - stakes and making transparent judgements based on the input. -
    + stakes and making transparent judgements based on the input. [1] +

    This framework is currently used by the U.S. Environmental Protection Agency. As our project intends to provide a robust solution for harmful algal blooms and therefore necessitates direct interaction with the environment, we must assess the long-term impacts of our project on the environment. The comprehensive - environmental assessment of our project focuses on the genetically altered bacteria Escherichia coli and the - potential impacts the altered bacteria may have on the environment. + environmental assessment of our project focuses on the genetically altered bacteria Escherichia coli + and the potential impacts the altered bacteria may have on the environment.

    Altered Physiology

    - Our project aims to develop E. coli that can degrade microcystins, a primary toxin that is produced by + Our project aims to develop E. coli that can degrade microcystins, a primary toxin that is produced + by harmful algal blooms. Our project involves two steps: detection of microcystins in water and remediation of said contaminated water. The first step, detection, involves using naked RNA to sense microcystins. Our team is aiming to create an aptamer-based sensor to recognize microcystins. The second step involves engineering - the mlr cassette into E. coli to breakdown the toxins. The mlr cassette is a series of six genes that gives - E. coli the ability to degrade microcystins, including mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF that each take + the mlr cassette into E. coli to breakdown the toxins. The mlr cassette is a series of six genes that + gives + E. coli the ability to degrade microcystins, including mlrA, mlrB, mlrC, mlrD, mlrE, and mlrF that + each take on different roles in breaking down the toxins. For instance, mlrD produces the genes responsible for transporting the microcystins into the cell. To carry out the degradation more efficiently, some mlr genes are designed to be transported to the cell’s periplasm. @@ -264,7 +265,8 @@

    In the event that our detection and remediation system gets integrated into drinking water treatment facilities, or is used for instantaneous lake testing and feedback, competition and biodiversity would be - the main concern regarding the modification of E. coli. However, our current system isolates the bacteria + the main concern regarding the modification of E. coli. However, our current system isolates the + bacteria from the freshwater ecosystems to avoid the risk of horizontal gene transfer. Regardless, our team has taken steps to ensure the containment of the modified bacteria by immobilizing them on alginate beads. Moreover, we are strategically placing filters in the inlet and outlet of the reaction chamber to ensure that @@ -277,7 +279,8 @@ lab. There does exist a possibility that the antibiotic resistant genes may be transferred to other bacterial strains. However, auxotrophy can serve as a potential solution to eliminate such genes. Auxotrophy is the inability of a gene to synthesize organic compounds needed for growth. By implementing auxotrophy - onto the altered E. Coli, we can decrease the possibility of the altered genes being transferred to other + onto the altered E. coli, we can decrease the possibility of the altered genes being transferred to + other bacterial strains.

    Gene Transfer
    @@ -296,24 +299,24 @@

    - The main environmental concerns about the modified E. coli genes would pertain to the possibility of the - mutated genes affecting the environment through gene transfer. Despite working primarily with the engineered - bacteria in a laboratory setting, our team has also designed filters in the reaction chamber to provide a - physical barrier, and the altered genes are treated by auxotrophy, a metabolic strain disabling them from - synthesizing organic compounds for growth, to make sure that they would not outcompete the wild strains. + The main environmental concerns about the modified E. coli genes would pertain to the possibility of + the mutated genes affecting the environment through gene transfer. Despite working primarily with the + engineered bacteria in a laboratory setting, our team has also designed filters in the reaction chamber to + provide a physical barrier that prevents the modified bacteria from entering the environment.

    Another crucial part of our project is the boat, whose main purpose is to gather samples from the lake and use GPS capabilities to record the coordinates of our sample collection sites. The boat also operates on an automated driving system that allows it to move to various GPS coordinates via motors. To ensure that no microplastics decompose into the water, the boat was made using plexiglass.

    - We have tested the boat on Cayuga Lake, and it is important to consider the laws regulating boat usage on - Cayuga Lake. According to the Tompkins County boat laws, it is permitted to drive a boat on the lake under + When looking at boat usage in areas, such as Cayuga Lake, it is important to consider relevant laws and + regulations. According to the Tompkins County boat laws, it is permitted to drive a boat on the lake under the speed limit, 5MPH within 100 feet of shore and 10 MPH between 100 feet and 500 feet of shore. Users are - also required to use enzyme-based cleaners to reduce the impact done on the water. We can program the boat - speed to be below the specified limit, so there would be no violation of law. Moreover, as the boat is - powered by batteries, there do not exist risks of potential oil spills and thus there is no need to use - cleaners to purify the water. + also required to use enzyme-based cleaners to reduce the impact done on the water. [2] Our boat is able to + be programmed to travel at a speed below the specified limit and runs no risk of a potential oil spill since + it runs on battery power, which eliminates the need to use cleaners to purify the water. + +

    @@ -335,7 +338,8 @@ project provides a novel post-contamination solution since effective long-term and short-term solutions are necessary to successfully mitigating the issue.

    - Although there is some concern that the altered E. coli used in our project may impact the environment and + Although there is some concern that the altered E. coli used in our project may impact the + environment and disrupt local ecosystems, we have put various safety constraints in place. These are mentioned in the CEA section, and our project takes great care to address these potential impacts on the environment.

    @@ -350,66 +354,129 @@ presence of microcystins. Most samples are collected by volunteers from local organizations, meaning collections tend to be free or inexpensive. After samples are collected, there are multiple ways to test for the presence of microcystins. The most common way is through an ELISA, which ranges from $125 to $200 - dollars per sample. The most common price for a test, however, is $125 per sample. There usually is more - than one sample so sending multiple samples will be costly. + dollars per toxin[1]. The most common price for a test, however, is $125 per toxin.

    Another variable in this analysis is the uncertainty of receiving results. From our conversation with Julie Lockheart from Owasco Lake Association, results from the samples take up to three weeks to process and - analyze. By this time, microcystins clear up and the results would not be effective in preventing damage - from the toxins. Our boat serves as a replacement for volunteers and collects samples based on areas in the - lake where HABs are most common. Although the boat costs around $200, it saves time by acting as an + analyze. By this time, microcystins degrade naturally and the results would not be effective in preventing + damage from the toxins. Our boat serves as a replacement for volunteers and collects samples based on areas + in the lake where HABs are most common. Although the boat costs around $200, it saves time by acting as an automated sample collector with high-resolution data capabilities. It can work throughout the day, mapping locations of blooms around the lake. The DNA aptamers with the gold nanoparticles serve to replace ELISA - kits and remove the issue of waiting for results. The gold nanoparticles cost approximately $150 for 25 mL - and the aptamer cost around $10. Our system is functional for 130 uses. This is significantly more - economical than the $125 per sample charged by laboratories to test using an ELISA. + kits and remove the issue of waiting for results. The gold nanoparticles cost approximately $150 for 25 + mL[2] and the aptamer cost around $10[3]. Our system is functional for 130 uses. This is significantly more + economical than the $125 per toxin charged by laboratories to test using an ELISA.

    At the moment, the price of clearing microcystins ranges widely. In a report done by the American Water Works association, depending on how long the treatment is, it could vary from $178 for 3 days of prevention - and treatment to $280,000 for 3 months of prevention and treatment of cyanotoxins. This is compared to our - project, which only requires $500 to build a small scale bioreactor that can last a significantly longer + and treatment to $280,000 for 3 months of prevention and treatment of cyanotoxins[1]. This is compared to + our project, which only requires $500 to build a small scale bioreactor that can last a significantly longer time.

    Damage caused by microcystins varies greatly depending on the water treatment facility and the amount of time the plant was exposed to the microcystins. One of the worst cases of cyanotoxins affecting drinking water was in Toledo, Ohio. The water plant in the city had to spend $54 million dollars on a new treatment - facility to remove contaminants like microcystins. This is, however, an extreme case, where Toledo was + facility to remove contaminants like microcystins[4]. This is, however, an extreme case, where Toledo was exposed to large amounts of microcystins for a long period of time. Regardless, this demonstrates the damage microcystins can do to water treatment facilities.

    The damage caused by microcystins can be negated by the early detection offered by the aptamer system. - Treatment would also be low-cost and effective. The bioreactor costs less than $100 taking into - consideration the alginate beads, the materials to make the actual reactor, and the engineered bacteria. The - alginate beads also last several years without degrading, so replacing the alginate beads would not be - costly. + Treatment would also be low-cost and effective. The bioreactor costs less than $500 taking into + consideration the alginate beads, the materials to make the actual reactor, and the engineered bacteria[5]. + The alginate beads also last several years without degrading, so replacing the alginate beads would not be + costly. Given the economic advantages of our system over existing methods of detection and remediation, our + product would allow for more cost-effective treatment of microcystin contamination to better protect + communities exposed to the dangers of HABs.

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    Law and Regulation
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    + [1] History of the Clean Water Act. (2017, August 8). + https://www.epa.gov/laws-regulations/history-clean-water-act. +

    + [2] HABHRCA. (n.d.). https://coastalscience.noaa.gov/research/stressor-impacts-mitigation/habhrca/. +

    + [3] Harmful Algal Blooms and Hypoxia Research and Control Amendments Act of 2011, November 13, 2012, 112-2 + Senate Report 112-237, Harmful Algal Blooms and Hypoxia Research and Control Amendments Act of 2011, + November 13, 2012, 112-2 Senate Report 112-237 (2013). +

    + [4] Governor Cuomo Unveils 12th Proposal of 2018 State of the State: Protecting New York's Lakes From + Harmful Algal Blooms. (2017, December 21). + https://www.governor.ny.gov/news/governor-cuomo-unveils-12th-proposal-2018-state-state-protecting-new-yorks-lakes-harmful-algal#_blank. +

    + [5] Harmful Algal Bloom (HAB) Action Plans. (n.d.). https://www.dec.ny.gov/chemical/113733.html. +

    + [6] Harmful Algal Bloom Action Plan Cayuga Lake, Harmful Algal Bloom Action Plan Cayuga Lake1–124 (n.d.). + https://www.dec.ny.gov/docs/water_pdf/cayugahabplan.pdf +

    + [7] 33 U.S. Code Chapter 53 - HARMFUL ALGAL BLOOM AND HYPOXIA RESEARCH AND CONTROL. (n.d.). + https://www.law.cornell.edu/uscode/text/33/chapter-53#. +

    + [8] Britannica, T. E. of E. (n.d.). Environmental Protection Agency. + https://www.britannica.com/topic/Environmental-Protection-Agency. +

    +
    +
    Comprehensive Environmental Assessment + (CEA)

    +

    + [1] Powers, Christina M., G. Dana, P. Gillespie, M.R. Gwinn, C. O. Hendren, T. C. Long, A. Wang, J. M. + Davis. “Comprehensive Environmental Assessment: A Meta-Assessment Approach.” Environmental Science and + Technology. Sep. 2012. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3439956/ +

    + [2] “Clean Up Your Act: Boat Clean!” Tompkins County Water Resources Council. 2007. + http://tompkinscountyny.gov/files2/planning/committees/WRC/documents/ItstheLawbrochure2007.pdf +

    +
    +
    Economic Analysis

    +

    + [1] “Laboratories that will accept samples for Harmful Algal Bloom (HAB) toxin analysis.” Environmental + Protection Agency. June 2016. + https://www.epa.gov/sites/production/files/2016-06/documents/hab_toxin_labs_list.pdf +

    + [2] “Gold nanoparticles.” Millipore Sigma. + https://www.sigmaaldrich.com/catalog/product/aldrich/741957?lang=en®ion=US +

    + [3] “DNA oligos.” Integrated DNA Technologies. + https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos +

    + [4] Schechinger, Anne. “The Huge Cost of Toxic Algae Contamination.” AgMag. June 2019. + https://www.ewg.org/agmag/2019/06/huge-cost-toxic-algae-contamination +

    + [5] “Sodium Alginate.” VWR. https://us.vwr.com/store/product/8887847/sodium-alginate +

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    Overview

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    Cornell iGEM understands the inherent risks of working in a lab facility and aims to take +all necessary precautions to ensure no personal or environmental harm occurs. To this +end, we have implemented the following safety procedures below. Our completed safety +form can be found here.

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    Specific Safety Concerns

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    Laboratory Safety - Our project involves regular use of ethidium bromide, a DNA- +intercalating agent known to cause cancer, as well as the use of powerful UV light, for +visualization of gel electrophoresis. We must prepare culture media with antibiotics, +which could be harmful to humans in large doses. We also work with ethanol lamps to +maintain a sterile environment, which do involve having an open flame on the benchtop.

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    Environmental Safety - If any biological materials escape from the lab there is a risk of +transfer of antibiotic resistance from our engineered strains into other organisms. The +ozone used to induce oxidative stress in plants could escape to the environment and +endanger animal and plant life.

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    Pathogens in Lab - We are mainly working with E. coli in the lab space. E. coli is found +in the human gut flora, but also has been widely used by iGEM teams for cloning +purposes and is categorized as Biosafety Level 1. We used K12 and B strains of E. Coli.

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    Microcystins - Microcystins are a Biosafety Level 2 toxin. It can damage the liver, cause +nausea, muscle weakness, and death. It has an LD 50 of 0.04 ug/kg if administered through +the intraperitoneal route, making it very dangerous to handle.

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    Safety Protocol

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    Wet Lab - All lab members wear nitrile gloves, closed-toe shoes, and use eye protection +when working with volatile chemicals or UV light. Gloves are replaced and hands are +washed immediately after using ethidium bromide or any of the metal solutions. +Members work in small groups to ensure if any harm comes to one, others are there to +assist. When working with a new reagent or piece of equipment, a faculty lab manager or +experienced member is always present to assist. +

    + There are taped off, designated areas for working with ethidium bromide. These areas are +cleaned before and after work and are the only areas the solution may touch. All toxic +waste is placed in a specialized receptacle and is picked up and disposed of by Cornell +Environmental Health and Safety. +

    + All disposables that come in contact with biologics are disposed of in biohazard waste. +The lab space also contains sharps containers for disposal of all sharps that contact +biological material. All biohazard waste is autoclaved and transported to the building's +centralized waste facility where it is disposed of as regulated biological waste. +

    + We maintain 2 copies of MSDS's for every chemical we use in the lab: one for our own +records and one for the lab manager and users of the lab space who are not part of our +team. The lab is equipped with flame-retardant benches, spill kits, safety showers, eye- +washes, and fire extinguishers. +

    + We worked with Cornell’s internal Biosafety Committee to develop an updated +Memorandum of Understanding and Agreement (MUA) as well as a set of Standard +Operating Procedures (SOP) for Microcystins, which can be viewed here. +

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    Dry Lab - We use the Emerson Machine Shop and the Rev facility for fabrication; each +of the dry lab subteam members has attended the prescribed training session for use of +the shop and has learned to use each of the tools safely. The dry lab subteam lead led +intensive trainings for our workspace including the 3D printer, drill, and other tools. Each +member of the dry lab subteam was also trained in the safe usage of the milling machine +and the metal lathe. +

    + All machine shop work is conducted under the supervision of the Emerson machine shop +or Rev staff. Safety goggles were worn at all times. Masks and gloves are worn as +appropriate. Closed-toe shoes and long pants were also worn when working in the +machine shop. While working in the machine shop we maintained a clean work +environment so we could maintain visibility at all times. When lifting heavy objects, +proper lifting technique was used, and an appropriate number of individuals were used +for lifting said objects. +

    + All members are required to clean up the office space after use, sweeping up sawdust and +putting away any tools used. Nothing in the office can obstruct the yellow lines taped off +to mark off a clear path through our workspace. Any items borrowed from one space +must be put back where they came from. +

    + A buddy system was to ensure the safety of members when they work in the lab spaces +during off hours. Any dangerous incidents that occur are taken seriously, and precautions +are implemented for future work. The team’s designated safety officer reviews all spaces +and informs the team each week if there are any possible unsafe situations that need to be +fixed. +

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    Training and Enforcement

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    Training - All team members who work in the wet lab must complete Cornell EH&S +general lab safety and chemical waste disposal courses prior to the onset of work. These +courses set specific guidelines and are the standard requirement for work in a biosafety- +level 1 lab at Cornell. Additionally, all team members must complete a lab orientation +session with the manager of the BME instructional lab, Dr. Shivaun Archer. During these +sessions, Dr. Archer familiarizes new members with the safety equipment and procedures +specific to the labspace in which we work. +

    + Prior to the onset of work for the year, all new members are required to go through a +safety training program. During this program, safety officers reinforce safety procedures +learned during the EH&S courses, discuss safety protocol pertaining to specific chemicals +with which we work, and ensure all lab members fully understand all safety procedures. +

    + To work with microcystins, members are also required to review the Microcystin SOP +and attend a Biosafety Cabinet training.

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    Safety Officers - The safety officers were team members chosen to directly supervise the +activities of the other team members. One team member each was chosen for the wet and +dry lab subteams to ensure that all team members were working safely, whether with +bacterial cultures or power tools. These team members also act as liaisons to the wet lab +and machine shop managers and, when necessary, the Institutional Biosafety +Committee to ensure proper equipment usage. +

    + These team members are responsible for discussing the proposed work plan for the +project with the wet lab and machine shop managers before starting work to ensure that it +is safe to continue. In the case of the wet lab in particular, this involves going through a +detailed list of protocols, including all organisms, chemicals, and genetic constructs being +worked with, to ensure conformity with the Environmental Health & Safety guidelines. +They must go through the same safety training as all other team members, but are +required to redo the training each time we recruit new members in order to keep up-to- +date with safety considerations. In addition, they maintain contact with the supervisors of +the workspaces, usually in the form of a weekly check-in, to discuss any safety concerns +that have arisen and ensure that equipment continues to be used properly.

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    Enforcement - Enforcement: Team members who violate safety rules are required to work under the +supervision of the safety officers for the remainder of the week, or until the safety officer +believes the member is capable of performing the task unsupervised. For multiple +infractions or complete disregard to safety protocols, a member may be restricted from +laboratory work until he/she undergoes EHS chemical safety online training again, and +demonstrates proper performance to a team leader of failed technique(s) in a controlled +setting.

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