How we integrated feedback from experts and stakeholders into our project to ensure that our project is responsible and good for the world.
Engaging Stakeholders: Overview
At the outset of project planning, Team SUNY Oneonta interviewed an expert in Harmful Algal Blooms (HAB), Dr. Kiyoko Yokota, to provide background information for our project with the aim of focusing our work on the goal of using cyanophages to help control HABs. However, as we began to research Cyanobacteria in depth, these goals began to shift toward Cyanobacteria engineering via Cyanophage gene delivery for the production of products such as plastics, proteins, biofuels, and more. With this objective shift, our team began to seek out experts who had experience working with Cyanobacteria and Cyanophages and their genetics. This led us to interview Dr. Andrew Millard and Dr. Stephan Klahn, both experts in these areas. They answered many of our questions and provided additional information regarding Cyanobacteria and Cyanophage. All correspondence with these three experts shaped and directed our project, helping us to rethink our objectives and our plan for execution of these objectives. More about their influence on our project is described below and in the summaries of our interviews and email correspondence, which are linked below.
In addition to integrating human practices into our synthetic biology work, we also incorporated human practices throughout the Collaboration, Partnership, and Education components of our project.
The Initial Track: Harmful Algal Blooms (HABs)
With the outbreak this summer of a large HAB in nearby Otsego Lake of Cooperstown, NY, our team wanted to learn more about these Cyanobacteria blooms, their toxicity, and what strategies are used today to control their spread. We interviewed Dr. Kiyoko Yokota, an Associate Professor of Biology at SUNY Oneonta. She is a limnologist with specific experience in Cyanobacterial bloom research and does extensive monitoring of the water quality of Otsego Lake. In our interview with Dr. Yokota, we learned Cyanobacteria come in many different sizes and morphologies. Some Cyanobacteria are so small, they are measured on the picometer scale. As such, these Cyanobacteria are hard to track since they cannot be imaged under a microscope and are most likely the most abundant. According to Dr. Yokota, the Cyanobacteria we would be interested in would be the larger strains. These form colonies and filaments, the scum that we so often see polluting our waters. Examples include the genus Dolichospermum and the genus Microcystis aeruginosa. These Cyanobacteria are mostly found in lakes and ponds, though they sometimes can be found in rivers. Some will produce cyanotoxins, but not all. We learned that these cyanotoxins can cause liver problems, harm the central nervous system, and irritate the skin. The toxins also irritate mucous membranes such as the eyes, sinuses, and mouth. Some incidents of exposure to these toxins have even resulted in death for both animals and humans. Dr. Yokota explained how Cyanobacteria thrive in high nitrogen and phosphorous environments, in warm weather, and with low wind disruption. This is why some preventative measures include reduction of nutrient build-up in bodies of water. Fertilizers and sewage often drain into lakes, building up as sediment at the bottom. By dredging the body of water, these excess piles of nutrients can be removed. According to Dr. Yokota, though, this method requires extreme care as the biological environment of the lake can be disrupted by dredging. Other methods include chemical binding of Cyanobacteria and phosphorous to cause the matter to settle at the bottom of the water. This is only a temporary fix, though, as the debris only settles for a finite amount of time, 5-10 years. Other methods include treatment with herbicides and algaecides. Even more treatment methods are being developed. Promising pilot studies have been done using sonication to break up Cyanobacteria cells and to then remove the disrupted scum by drawing it up from a barge. The results for these new studies are mixed, though.
After our interview with Dr. Yokota, our team began to do more research on Cyanobacteria. We found evidence of Cyanophages being responsible for Horizontal Gene Transfer in Cyanobacteria (1,2). This proved very interesting to us, and we began to study the matter in detail. This resulted in an objective shift in our project, a shift toward experimenting with Cyanophage Gene Delivery.
Objective Shift: Cyanophage Gene Delivery
As our project objective shifted toward Cyanobacteria engineering via Cyanophage gene delivery, our team realized the need for expertise in the area of Cyanobacteria genetics. In our search for experts, we came across Dr. Andrew Millard, an Associate Professor at the University of Leicester, United Kingdom and Vice President of the International Society for Viruses of Microorganisms. He has had years of experience working with Cyanobacteria and Cyanophages as well as extensive experience in all kinds of bioinformatics work. For example, he has worked on marine Cyanophage characterization, microbial bioinformatics, bacteriophage bioinformatics, phage-host interactions, and more.
In our initial email correspondence with Dr. Millard, he explained that tagging the capsid protein of our Cyanophage with biotin would not affect development of the capsid. In regard to our question whether we should use the Cyanophage Syn9 or S-TIP37, he explained that the hardest part of our project would be culturing the Cyanobacteria, therefore phage type would be of lesser importance. As we questioned him about specific genes of the phage that allow for the recognition and binding of a Cyanobacteria host, he shared with us that there has not been much success in identifying which proteins are responsible for these interactions, and that he could only give a good guess at which proteins these were. He also shared with us many details concerning the culturing of Cyanobacteria. The process is very tedious, he explained, and Cyanobacteria are very sensitive to outside influences such as light and temperature. Therefore, maintaining constant temperature and light are key to avoid killing your culture. Changes in temperature and light can be as long as they are gradual. Dr. Millard also explained the patience needed while culturing Cyanobacteria, as the colonies double in approximately 24 hours.
After initial correspondence via email with Dr. Millard, our team performed some bioinformatics work to determine which phage we would use. As time progressed, we wanted to meet with Dr. Millard to receive expert feedback on our progress, so we set up a virtual meeting with him. He suggested that if we tag our capsid protein with biotin, we should attach it to the C-terminus, as this had previously been accomplished for a T7 Bacteriophage. He also explained that designing our phagemid for expression in E. Coli was a good idea, as this is commonly done for other phages. Most importantly, he confirmed with us that a T7-like phage would be better to start phagemid design with rather than a T4-like phage, as T4-like phages are large and complex.
After meeting with Dr. Millard, we wanted to seek out a professional currently working in gene delivery and engineering of Cyanobacteria to determine what the benefits and drawbacks of phage gene delivery would be. In our search for such an expert, we came across Dr. Stephan Klahn, group leader of Molecular Biology of Cyanobacteria for the department of Solar Materials at Helmholtz Centre for Environmental research in Germany. Most of Dr. Klahn’s publications over the past four years have been related to Cyanobacteria, and many of these specifically detail genetic engineering of Cyanobacteria. With this repertoire, Dr. Klahn proved to be the perfect candidate for questions regarding phage gene delivery. Therefore, we emailed him a list of questions. In response, Dr. Klahn explained that he is in the final stages of publishing a book, The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories (3). He shared the final proof with us which detailed the common gene transfer methods used in Cyanobacteria engineering today. In summary, there are many gene transfer methods used. Two of these are transconjugation and electroporation, and many strains of Cyanobacteria are naturally competent. However, Dr. Klahn explained that not all Cyanobacteria can be transformed by the aforementioned methods, and not all are naturally competent. Thus, if our team was able to develop a phagemid that could be used in the future to transform these more difficult strains of Cyanobacteria, the advancement would be a great help to synthetic biologists. This word from Dr. Klahn motivated our team, as the idea of advancing self-sustaining production methods is a passion of ours.
Dr. Yokota, Dr. Millard, and Dr. Klahn each contributed immensely to our project, shaping its path with information, advice, and constructive criticism. To each of them we say, thank you for your investment in our project. We are forever grateful for the expert insight you shared with us.
Aminov R. I. (2011). Horizontal gene exchange in environmental microbiota. Frontiers in microbiology, 2, 158. https://doi.org/10.3389/fmicb.2011.00158
Bailey, S., Clokie, M. R., Millard, A., & Mann, N. H. (2004). Cyanophage infection and photoinhibition in marine cyanobacteria. Research in microbiology, 155(9), 720–725. https://doi.org/10.1016/j.resmic.2004.06.002
Opel, F., Axmann, I.M., Klähn, S. (2022). The Molecular Toolset and Techniques Required to Build Cyanobacterial Cell Factories. In: Advances in Biochemical Engineering/Biotechnology. Springer, Berlin, Heidelberg. https://doi.org/10.1007/10_2022_210