Team:UC Davis/Human Practices

Human Practices

  • In our efforts to best understand how we can provide an ethical and useful contribution, as well as ensure our project is reliable, and reproducible, we met with professionals from the biotech industry, the government, the field of bioethics, and the field of journalism. We then integrated the suggestions of these professionals into our project wherever we could.

Community

  • Ethics

      Bioethics

    • Mark Yarborough: Dr. Mark Yarborough is a Bioethics professor at UC Davis working as the Dean’s Professor of Bioethics.

      • Why did we approach them?

    • Dr. Yarborough has spent much of his career in the field and practice of bioethics, 10 of which were at UC Davis. He also has worked as an advisor for past UC Davis iGEM teams, so he seemed like the perfect person to advise us on ethically sound project design.

      • What did we learn?

    • Our meeting was fruitful as Dr. Yarborough introduced us to concepts that would ultimately make our project reproducible and in line with bioethics.
    • We also learned that some antibiotics save time in the trial phase by having deep and accurate analysis done on them in the lab research phase. If we do a thorough job of characterizing our BGCs and their secondary metabolites, the eventual production of new antibiotics could come sooner rather than later.

      • Computational Ethics

    • C. Titus Brown: Dr. C. Titus Brown is an associate professor at UC Davis working on development of the khmer software, for faster and more efficient sequence analysis of high-throughput sequencing data.

      • Why did we approach them?

    • Dr. Yarborough suggested that in order to truly understand the specifics of computational ethics we should speak to someone experienced in the field. This led us to bioinformatician, Dr. C. Titus Brown.

      • What did we learn?

    • Reproducibility is important for any scientific project, but Dr. Brown stated that reliability is just as important. We learned of ways to make our program and computational design more intuitive for future users.
  • Government

      JGI

    • The Joint Genome Institute is a part of the Department of Energy, which performs research in the production of useful substances like biofuels.

      • Why did we approach them?

    • JGI boasts a database of hundreds of partially annotated fungal genomes. Their scientists, Dr. Grigoriev and Dr. Salamov, were generously open to the idea of allowing UC Davis to explore these genomes in the pursuit of new biosynthetic parts.

      • What did we learn?

    • We gained some knowledge about secondary metabolite regulation in biosynthetic gene clusters, as well as the importance of public databases for researchers.
  • Industry

      Novozymes

    • Novozymes is a global provider of biological solutions, specifically in relation to enzyme production. We met with Dr Amanda Fischer, a staff scientist working with filamentous fungi, to gain valuable input for our project.

      • Why did we approach them?

    • Since novozymes is a leading synthetic biology company, we wanted to gain knowledge from the industry about improving our experimental design. We were also curious about what sort of work is required in an industry-level project, as opposed to an academic project.

      • What did we learn?

    • We learned about specific ways to work and communicate with our community to ensure that our project is understood and trusted. We also learned about potential ways to optimize our chassis for industrial production.

      • Marrone BioInnovations

    • We met with Dr. Vasavada, senior VP of R&D and chief tech officer, as well as Dr. Brittany Pierce of Marrone BioInnovations. They have a deep understanding of the issues involved with research and production of industrial fungal strains.

      • Why did we approach them?

    • The employees at Marrone BioInnovations are pioneers for bio-based pest management and plant health. We approached them to inquire about their quality standards for projects, considering the tremendous work they do with filamentous fungi.

      • What did we learn?

    • They provided us with insightful knowledge about some challenges of working with filamentous fungi. They also encouraged us to look closely at our proposed gene insertions to ensure compatibility with our organism. In addition, we learned ways to optimize production from a chassis:
      1. Modify production line instead of organism. Tailor your production scheme to best fit the organismal chassis.
      2. Create teams in charge of optimizing specific parts of the procedure. Create an “assembly line.” i.e. a fermentation team to scale up and produce efficiency, a chemical team for selecting reagents, and a formulation team for stability of active compounds.
  • Outreach

      Communication

    • Trina Kleist is a national award winning journalist with 34 years of journalism experience. She is pursuing a masters degree in Science Communication and Media Innovation and expected to graduate in December of 2020.

      • Why did we approach them?

    • As an experienced journalist, Ms. Kleist is skillful at communicating with people. She also communicates with the goal in mind of gaining useful information, and delivering her own point of discussion. We hoped to demonstrate some of this skill in our own science communication presentations.

      • What did we learn?

    • Ms. Kleist gave us valuable insight in regards to our science communication outreach, and how to better engage our audience so that they would have something significant to take away from the presentation.

    • Integrated Human Practices

    Ethics

      Bioethics

    • Our interview with Dr. Yarborough shifted our collective mindset towards one of ethics and human practices. We were advised to look ahead to understand what unforeseeable consequences our project might have in the future, and take steps to mitigate those issues. Increasing accessibility of fungal parts could lead to discovery of new toxins which, if not used carefully, could be harmful to people or the environment. Mitigating this risk comes down to understanding which model organism we should use, and how to use it safely.
    • Dr. Yarborough also emphasized to us the concept of reproducibility. Reproducibility is very important in science across the board. However, making a project reproducible in bioinformatics requires a different approach than in synthetic biology. To implement reproducibility in our experimental design, we created a Procedural Handbook. This allows future researchers to copy our culturing protocol if they wish, saving them the work of digging through literature.
    • Specifically for bioinformatics, we were counseled to be aware of how many software errors go unnoticed in the making of a program. To help us prevent this, Dr. Yarborough referred us to Dr. C Titus Brown, who has written extensively about these errors[1]. Dr. Brown shared his insight on ethically developing a program.

      • Computational Ethics

    • Our meeting with Dr. Brown was enlightening to say the least. One of Dr. Brown’s mantras that stuck with us is as follows: organization is the key to a reproducible program. We learned that extensive documentation, and creating a log of the evolving code (also known as “version control”) grants accountability and a formatted explanation of the code’s beginning and end [2][3][4]. We took this advice, and implemented it in our own software by adding extensive documentation to our GitHub repository. We also developed a visual representation of our workflow for outside users of our software to familiarize themselves with how it works.
    • Besides reproducibility, Dr. Brown also largely emphasized reliability. He charged us with having as many people test our code as possible, in order to ensure results are constant and reliable. So, not only is our software tested by members of Spore_Core; we also made our GitHub repository public so that people can continue to test the software for bugs and errors. This helps optimize the program for all users.

    Integrations

    1. Clear Software Instructions
    2. Procedural Handbook

    Government

    • The Joint Genome Institute has a public database of hundreds of fungal genomes which are available to any researcher. This database is called mycocosm, and it boasts a website with video tutorials to help navigate the site’s many functions [5]. This level of public availability is an aspect we strive for in our own human practices. We provide a public github with tutorials (both written and in video) for using our software tools.
    • JGI provided us with 664 filamentous fungi genomes from their database. We met with them early in the project, once it was clear that we would be limited to computational methods. Dr. Salamov and Dr. Grigoriev of JGI advised us to use filamentous fungi genomes in particular, because many of them have not been fully annotated. The lack of data for these fungi in academia remains an issue [6]
    • The biosynthetic gene clusters (BGCs) of these genomes were predicted by JGI based on common, easily recognizable BGC genes (ie. protein kinase genes, non-ribosomal peptide synthase genes, and terpene genes) in close proximity to each other. The promoter regions that we use were defined by the same terms. Since we began sifting through these genomes to discover binding sites, JGI has remained in contact with us via email. We are very grateful to Dr. Grigoriev and Dr. Salamov for their contribution towards this iGEM project.

    Integrations

    1. Tutorials
    2. Public Github

    Industry

    • Dr. Fisher provided us with excellent knowledge to implement into our project. She supported our decision to make Aspergillus Niger our chassis organism, stating that it is “tame and sporulates easily.” One issue that we had discussed prior to the meeting was the possibility of accidentally creating a resistant strain by transforming antibiotic resistance markers (such as hygromycin) into our chassis via E. coli. Dr. Fischer informed us that preventing this requires excision of that selection marker gene before production, making it inadequate for industrial use
    • However, as our project only aims to confirm binding of our prospective transcription factors through expression of a reporter gene, and not to produce a product, we decided to continue with the antibiotic marker. However, if our chassis is eventually used for industrial production of secondary metabolites, we would need to modify our procedure by using dominant selective markers, rather than antibiotic resistance genes. An example of such dominant selective markers is amdS, a gene used in one of our plasmids, which allows for selective growth in the presence of acetamide [7]. Markers such as this are recommended for industry purposes.
    • We also picked Dr. Fisher’s brain about investor relations. We asked, “How do you effectively communicate your product usage to the community for them to accept it as useful/good?” She answered that the use of a “minimum viable product”, or a demonstration of how the product works, along with a collaborative effort in which the customer participates in product development, is essential for community acceptance. She called this an “agile” approach. Since our project aims to expand the filamentous fungi catalog for future researchers, we consider our continued work alongside JGI to be our “agile” approach, and our list of discovered binding sites to be our “minimum viable product”.
    • Marrone BioInnovations had some first hand experience working with aspergillus. They explained some of the outstanding issues involved in working with filamentous fungi, and why it is such an understudied field. One of the most prominent of these issues is that it is difficult to sequence and annotate the genome. They echoed the concerns of Dr. Salamov and Dr. Grigoriev of JGI about the lack of data, and added that the data that exists tends to be non-predictive. They also mentioned a lack of effort in this particular group of organisms, with greater focus going toward mammalian cells. In addition, there are private companies studying filamentous fungi, who seldom share their information. With this in mind, we aim to make our results accessible and reproducible, in the hopes that other researchers can utilize our tools to expand the catalog of filamentous fungi.
    • They also encouraged us to make sure that our reporter gene was compatible with our organism. We originally had the idea of using standard GFP as our reporter gene. However, after an extensive literature search, we happened upon a reporter gene with more promise. We discovered and implemented sGFP, a variant of GFP, where a serine at position 65 is replaced with threonine[8-9]. This small change results in brighter fluorescent signals in filamentous fungi.
    • Lastly, we posed the question of whether or not it would serve us well to transform multiple plasmids at once into our organism to save time. Dr. Pierce urged us against this strategy, because the rate of recombination in a transformation is already low. Therefore the odds of retaining two plasmids in a single cell at once would be miniscule. We heeded their advice, and wrote our protocol to include two separate transformations, using different selectable markers.

    Integrations

    1. Use dominant selective markers for industrial secondary metabolite production, rather than antibiotic resistance genes
    2. Agile approach
    3. Optimized reporter gene
    4. Consecutive Transformations

    Outreach

    • Effective communication stems from clarity, specifically clarity in our own thoughts. We were encouraged to create a “lead”: a short hook sentence with the 3 most important topics we wanted to get across. This was implemented in our presentations to high school and junior college students. The three topics were clear: bioinformatics, synthetic biology, and iGEM. Ms. Kleist also suggested telling a story to help relate our topic to the students’ lives. We implemented this idea at Nevada Union High School, and it seemed to make a difference in the engagement of the students.
    • Another tool to keep students engaged is adding something fun. Especially with high schoolers, it seems to be a great way to “close the loop” on the topics of our presentation. We created a Kahoot game-- which is a speed-based, competitive multiple choice quiz-- so all the participants could test their knowledge. In this we illuminated important topics and key concepts that we wanted them to remember.
    • Lastly, Ms. Kleist suggested ending the presentation with a call to action, something to give them a sense of urgency and encouragement. Ultimately, we wanted them to be excited enough about the presentation to form their own iGEM team. The call to action was short and sweet, explaining that other high school teams participate in iGEM, and that research like ours is not an unrealistic goal.
    • In order to diversify our outreach, we took to social media. However, Ms. Kleist heavily suggested getting someone with a bigger following to do the “heavy lifting” for us. We reached out to the Bio Innovation Group at UC Davis and they agreed to post our promotional video and instagram handle to their weekly newsletter. With this we hope to reach a larger community that can learn about synthetic biology opportunities. We currently use Instagram for anyone interested in getting to know the team or our project. We also have a YouTube channel with our team’s video media. You can find these at the following links:
  • “Imagine that in 20 years, air pollution has gotten so bad that people can hardly breathe. Synthetic Biologists could come up with a way for an organism to take in pollution and spit out something cleaner, or they could develop a way for our bodies to intake the harmful chemicals and survive. The applications are endless with the only limiting factor being your imagination.”
  • “In 10 years maybe there will be needs for gene modification in crops to better accommodate rising temperature, and maybe there will be more demand for an enzyme that can convert saltwater to freshwater. These issues are waiting to be solved by YOU. ”

    • References

    • [1] Soergel, David A. W. “Rampant Software Errors May Undermine Scientific Results.” F1000Research, vol. 3, 2015, pp. 1–14, doi:10.12688/f1000research.5930.2.
    • [2] Reiter, Taylor, et al. Streamlining Data-Intensive Biology With Work Ow Systems. 2020.
    • [3] Brown, C. (2020, April 20). Living in an Ivory Basement Stochastic thoughts on science, testing, and programming. Retrieved October 24, 2020, from http://ivory.idyll.org/blog/2020-software-and-workflow-dev-practices.html
    • [4] Perkel, Jeffrey M. “Challenge to Scientists: Does Your Ten-Year-Old Code Still Run?” Nature, vol. 584, no. 7822, 2020, pp. 656–58, doi:10.1038/d41586-020-02462-7.
    • [5] Grigoriev, Igor V., et al. “MycoCosm Portal: Gearing up for 1000 Fungal Genomes.” Nucleic Acids Research, vol. 42, no. D1, 2014, pp. 699–704, doi:10.1093/nar/gkt1183.
    • [6] Meyer, Vera, et al. “Current Challenges of Research on Filamentous Fungi in Relation to Human Welfare and a Sustainable Bio-Economy: A White Paper.” Fungal Biology and Biotechnology, vol. 3, no. 1, BioMed Central, 2016, pp. 1–17, doi:10.1186/s40694-016-0024-8.
    • [7] Solis-Escalante, Daniel, et al. “AmdSYM, A New Dominant Recyclable Marker Cassette for Saccharomyces Cerevisiae.” FEMS Yeast Research, vol. 13, no. 1, 2013, pp. 126–39, doi:10.1111/1567-1364.12024.
    • [8] Chiu, Wan-ling, et al. “Engineered GFP as a Vital Reporter in Plants.” Current Biology, vol. 6, no. 3, 1996, pp. 325–30, doi:https://doi.org/10.1016/S0960-9822(02)00483-9.
    • [9] Felitti, S., et al. “Papers in Plant Pathology Green Fluorescent Protein Is Lighting Up Fungal Biology Green Fluorescent Protein Is Lighting Up Fungal Biology.” Society, vol. 67, no. 5, 2015, pp. 1987–94, doi:10.1128/AEM.67.5.1987.