Poster: CCA_San_Diego

Acefate: Degradation of the Pesticide Acephate Using Genetic Modification

Abstract: The bio-degradation of the insecticide acephate using genetically modified Escherichia coli is studied in this project. Acephate exposure causes severe environmental and human side effects as well as paralysis or death. In this study, we enriched samples of acephate-treated soil for 5 weeks to propagate the growth of acephate-degrading bacteria. 10g from each sample was diluted 10-fold. DNA was subsequently extracted from bacterial colonies using the genomic etNA extraction and the 16S rRNA gene was sequenced. Degradation was monitored with HPLC and LC-MS. Certain genes, such as OPD, were isolated and analyzed using bioinformatics tools including CDD. Amplified genes were inserted into base plasmid pMMB206 using restriction digest protocol into E. coli K12. BetI, a transcription factor-based kill-switch, was added to the plasmid. While experiments could not be performed, MATLAB modeling and literature indicate the approach provides an effective pathway, with >99% degradation of acephate and all harmful intermediates.

Team: Our team consists of 22 members (ALL from Canyon Crest Academy, a public high school in San Diego, California) who are all passionate about synthetic biology! We have many interests, including swimming, karate, earth science, robotics, and more, but it was iGEM that brought us all together.

Team Leaders: Andrew Gao, Mason Holmes, and Ayush Agrawal

Team Members (in no particular order): Amogh Chaturvedi, Noah Zhang, Andrew Sun, Anny Wang, Sid Udata, Andrew Kang, William Kang, Chris Jung, Nathan Robinson, Grace Wang, Jessica Lin, Makenna Holst, Joanne Lee, HanMin Kim, Ella Adams, Dharmik Grandhi, Archit Chaturvedi, Dylan Feldstein, Natalie Feldstein.

Attributions: Outside of our team members, we would like to thank our wonderful CCA science teachers; all four iGEM teams that we collaborated with; the CCA Foundation for financial advice and fund management; Inovio and Qualcomm for financial support; as well as all of the professors, farmers, government officials, and graduate students who helped out with our lab, modeling, and human practices throughout the journey.

Introduction

Inspired by environmental issues plaguing our community, this year we sought to tackle the issue of acephate contamination. Acephate is a toxic pesticide that is widely used, kills wildlife, and has health effects on humans.

Over the course of the season, our team approached the problem in many ways. We developed an extensive flexible lab protocol with adaptable measures, modeled acephate degradation, consulted hundreds of community members, spoke with farmers, and more.

We:

developed lab protocol with fall back measures
performed degradation modeling on acephate
spoke with 245 diverse community members
interviewed farmers and stakeholders
consulted 40+ experts
developed plasmids and kill switch
created engineering success plan in case something goes wrong with lab
contributed literature findings to existing parts
meaningfully collaborated with DNHS, KSA Korea, Lambert_GA, and PYMS_GZ_China 2020 iGEM Teams
developed a 20 page survey handbook for iGEM teams
developed a 57-page curriculum and spread it internationally online via social media

NOTE: Because of COVID-19 shutdowns, we were not able to access a lab this year. All of our lab protocols, therefore, are purely hypothetical for now.

We plan on experimentally validating our project in the future, as well as improving our proposed implementation.

Problem and Solution

Acephate is an insecticide commonly used on crops and in places such as golf courses. While it is useful in managing pests, acephate has many negative effects on human health and the environment. Because of its wide use, acephate is a far-reaching global issue.

Acephate is a potential carcinogen, causes dermal abnormalities and brain damage, and has fetotoxic effects. In the environment, it is responsible for the deaths of bees, fish, and birds. Acephate causes all these effects when it leaches into the ecosystem through soil, rivers, and more.

We were initially inspired to tackle pesticide runoff after a school science project. We had collected water samples from our local river and discovered very low diatom diversity, indicating some sort of water pollutant. After research, we discovered that there were golf courses and well-kept fields upstream of the river. We also found that water pollution by pesticides and chemicals is a prominent issue in our community.

In 2000, the USDA reported that acephate was used at a rate of 40% of all insecticide use, making it the most used insecticide, in which organophosphates make up 34% of insecticide sales. Hence, our team narrowed our focus to solve the problem of acephate.

An ideal decontaminant candidate must be able to rapidly detoxify the agents at a molecular level and be able to decontaminate a variety of surfaces such as paint, concrete, rubber seals, asphalt, metal, plastics, clothing, and skin. Additionally, these reagents should be environmentally friendly without lasting detriment to soil, vegetation, animal life, or underground water sources.

Our solution is to engineer genetically modified bacteria that can degrade acephate into harmless byproducts. The bacteria will be applied to fields via a spray and have an integrated killswitch to ensure safety in implementation. For more technical details, please see our Lab Overview section.

Human Practices

HP/IHP highlights

245 person survey with 7,000 word analysis
Conversations with 40+ experts
Interviews with farmers and agriculture experts
Directly inspired implementation, science communication, and lab.
Created 20 page iGEM survey guide for teams to help them make better surveys (on wiki under contributions)

Researcher interviews/emails

Emailed government officials, sanitation workers, farmers, bee keepers, professors, and undergraduate and graduate students involved in the field
Got 40+ insightful emails that caused us to change our lab protocols, modeling, and our overall design. Our project was a human-centered design and HP led the way of our project
Did thorough analysis on each email from professionals and communicated with our team to ensure the proper changes were made

Farmer interviews

Had interviews with international farmers from China and India, who are directly impacted by acephate pollution.
In fact, Pankaj Mandloi, the Indian farmer actively uses bacteria in the soil that form positive interactions and symbiotic relationships with the plants to ensure the maximum natural growth of his crops. His opinions were heavily factored in our product
The farmers gave us the general consensus that a bioreactor is infeasible and more of a pain than it is beneficial. They helped us customize our product for our end users which are the farmers. We switched to a spray method; but to still make sure we were meeting the public's concern for safety we added a killswitch.
We had an interview with Warren Bacon, Agriculture Standards Inspector at County of San Diego. He gave us insights on the governmental as well as safety aspects of our project, he also gave us contacts to other high level government servants who gave us further knowledge and insights.

Survey analysis

Conducted a survey that measured the participants’ knowledge and comfortability with our project (and related topics), as well as asked for any suggestions to be integrated into our final project.
We found that there were participants of all age groups, education levels, and gender, so that results best reflected the opinion of the general public
We found that in general the participants had high approval ratings (an average of 6.99 on a scale of 1-10 regarding comfortability with GMO products) for our project involving GMO products because it benefited the world, but would be more comfortable with the project if it conducted in an isolated environment (91.1% or 216/237 of the responses would be comfortable with or more comfortable with our experiment if it were conducted in sealed off tanks) and went through extensive testing.
The survey also indicated that most participants believed harmful pesticide residues are very prevalent in soil (on a scale of 1-10, the prevalence of soil residues was on average 7.88), and that pesticide use is harmful but necessary (61.33% (138/225) of the responses indicate that pesticides are harmful but necessary), which led us to believe our solution to biodegrade the pesticide was the ideal solution (still allows the use of pesticides while reducing its harmful side effects)
In our correlation analysis, we determined that there was a significant positive correlation between the level of knowledge of our participants and their comfortability with our project, meaning that professionals/informed individuals would be more likely to approve of our experiment.
Shows that people who are aware of all the pros and cons of our experiment would be more likely to be comfortable with it, indicating that our project is overall beneficial to the world

How HP affected our project: At first, we considered using a bioreactor, a large tank where bacteria and soil would be stored. However, our interviews with farmers revealed that a bacterial spray would be much more feasible. Taking concerns about safety into consideration, we decided to implement a kill switch which will kill the bacteria after the acephate is degraded.

After our HP team did an analysis based on our survey results it revealed to us that the survey respondents that were more educated in synthetic biology were more accepting of synthetic biology projects. This discovery led us to create the Science Communication team which promotes science education in our case educating people more about the basics of synthetic biology.

Science Communication

We hosted a summer camp that allowed middle school and high school students to learn topics pertaining to the fields of synthetic biology and biotechnology.

We distributed our material via a 57 page document, social media services, and through video-sharing networks

We translated our document into Korean, Chinese, French, and Spanish, which allowed students from different parts of the world to learn from our document. We also performed text-to-speech on our material, which allowed students with visual impairments and disabilities to use our material. Team KSA-Korea aided us in editing the Korean translation of our document.

Finally, we designed a survey to analyze the quality of our materials through a pre and post survey between 95 individuals. People of all ages generally improved by 3.9/10 points after looking through our materials, and 87 % of participants found the materials helpful.

Lab Overview

NOTE: Because of COVID-19 shutdowns, we were not able to access a lab this year. All of our lab protocols, therefore, are purely hypothetical for now.

First, we sampled soil treated with acephate for over ten years and enriched our sample to grow out acephate-degrading bacteria. We then performed serial dilutions to isolate our bacteria and plated our isolated strain. Our next step was 16s rRNA Sequencing. We separated DNA from the bacteria and sequenced it with universal primers 27f and 1429r. We then analyzed bacteria with HPLC + LC-MS to confirm that our strain could degrade acephate. PCR was run to identify genes in isolated bacteria and to find a possible OPH degrading gene using Conserved Domain Database primers. To maintain environmental safety, we constructed a kill switch. This would trigger bacterial death after acephate sufficiently degrades.

Modeling

Why modeling?

1.Estimate the amount of time needed for the pathway to go to completion and acephate to be degraded below levels deemed safe by the EPA

2.Ensure that all major harmful intermediates (i.e. methamidophos) degrade to safe concentrations, ensuring that our process has no unintended byproducts

Reaction pathway in MATLAB SimBiology.

2.Methamidophos and DMPT (intermediates), while not explicitly mentioned by the EPA, are also estimated to be under their safe concentrations around 900 hours (~38 days).

Differential equations used in modeling with corresponding rate constants.

Plasmids

To enable acephate degradation, our amplified degrading gene was transformed into E. coli. OPH, our candidate gene, degrades Acephate as it can hydrolyze P-O, P-F, and P-S bonds in organophosphate compounds. The restriction enzymes BamHI and EcoRI were used in a standard restriction-digest transformation for OPH.

Our killswitch was modeled after a 2016 study that designed “toggle switches” to kill bacteria in the reduced concentration of a signaling molecule. It works by switching the bacteria into a “survival” lacI state and a “death” TetR state coupled with toxin expression. For our project, we used Betaine inhibitor, or BetI, a transcriptional regulator from the same TetR family. BetI uses choline as an inducer in a pathway connected to acephate degradation. Thus, with reduced acephate and choline concentration, our bacteria is switched into a BetI state, where induced toxin expression regulated by the mf-lon protease effectively kills the bacteria.

Engineering Success

To ensure that our experiment was successful, we designed our lab procedure to be flexible and account for errors. Starting from enrichment, we will make sure to use proper sourcing and isolation techniques, and enrich in variable environments with different pH and temperature to generate diversity in isolated strains. As we are searching for our candidate gene in a potentially novel bacteria, the specific primer sequence may vary. In that case, we will use bioinformatics tools such as the Conserved Domain Database and consider other genes involved in degradation. However, may acephate degrading genes have been shown to yield highly specific sequence similarity across species.

We will also maintain proper lab practice, such as testing multiple trials, proper storage, saving controls and backups, to optimize our transformation in the case of error. The insert:vector ratio is variable in our procedure because of different degrading genes: we will work around our plasmid construction to optimize this ratio and choose appropriate restriction enzymes. Regarding our kill switch, we are unable to foresee the validity of our proposed BetI pathway, as it has not been experimentally proven to account for endogenous soil concentration or the enzymatic bacterial pathway. In the case our proposed pathway does not work, we will consider other transcriptional regulators from the same family or consider other bio-containment methods.

Collaborations and Partnerships

Collaborations:

We had a collaboration with PYMS_GZ_China where we helped give them judging feedback (they are a first-year team) as well as helped them with their lab protocol and in science and communication. They helped us out without lab protocol, IHP, and suggested flowcharts and diagrams in our presentation.

Another collaboration we participated in was with Lambert_GA. They assisted us with modeling and both teams shared their projects and gave feedback to one another.

Partnerships:

We had a partnership with Del Norte High School where we had 12 total meetings that spanned for 45 minutes each. We gave each other constructed feedback each week, exchanged surveys for wider distribution, and brainstormed plans of action for fundraising and projects and meetups. CCA helped DNHS with modeling and IHP, and gave them extensive feedback on their survey.

Our second partnership was with team KSA_KOREA. We met every week on Saturday for 30 Minutes since August 10th. We traded surveys and made environmental analyses of each other’s projects. CCA also received key mentorship from one of KSA_KOREA’s advisors, Yoon ki Joo on modeling and our lab protocol.

Parts, Contribution, and References

Below is the list of parts we modified, as well as a summary of the information we contributed for each.

We created an original 20 page comprehensive guide to iGEM surveys in order to help teams create good, useful surveys. A member of the iGEM HP Committee approved of our guide. The guide gives useful information on ethics, consent rules, types of questions, data analysis, and more.

References

Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nature chemical biology, 12(2), 82–86. https://doi.org/10.1038/nchembio.1979

Lin, Ziqiu, et al. “Degradation of Acephate and Its Intermediate Methamidophos: Mechanisms and Biochemical Pathways.” Frontiers in Microbiology, vol. 11, 2020, doi:10.3389/fmicb.2020.02045.

Nivetha, M. & Linnett, N. Isolation and Determination of Efficacy of Acephate Degrading Bacteria from Agricultural Soil. Journals Iosr (2015). doi:10.6084/M9.FIGSHARE.1347502.V1

Pinjari, A., Pandey, J., Kamireddy, S., & Siddavattam, D. (2013). Expression and subcellular localization of organophosphate hydrolase in acephate-degradingPseudomonassp. strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides. Letters in Applied Microbiology, 57(1), 63-68. doi:10.1111/lam.12080

Ramu, S. & Seetharaman, B. Biodegradation of acephate and methamidophos by a soil bacterium Pseudomonas aeruginosa strain Is-6. Journal of Environmental Science and Health, Part B 49, 23–34 (2013).

Yao J, Zhu YC, Adamczyk J, Luttrell R. Influences of acephate and mixtures with other commonly used pesticides on honey bee (Apis mellifera) survival and detoxification enzyme activities. Comp Biochem Physiol C Toxicol Pharmacol. 2018 Jul;209:9-17. doi: 10.1016/j.cbpc.2018.03.005. Epub 2018 Mar 18. PMID: 29563044.

Team:CCA San Diego/Poster

Poster: CCA_San_Diego