Team:Stony Brook/Integrated.html
From the very beginning of our project, we have prioritized reaching out to professionals for their opinions on our work. Even before we finalized our project, we knew that gaining insight from others would be a detrimental part of our work. Talking to farmers and scientists and listening to their advice has shaped our research and design as well as how we use our platform to bring awareness of synthetic biology.
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After coming up with our first design idea, which revolved around the use of Bacteriorhodopsin (bR), we met with our advisors to discuss its feasibility. Dr. Rest quickly explained to us that manipulating bR insertion would influence strong selective pressure for mutations to remove that function. Even if we introduce this system, we may have a difficult time making sure that this construct remains in the organism. Additionally, we would have to ensure that we implement a way to prevent mutations during the propagation that removes it. This would be done by including some part that is absolutely necessary for the organism to survive so it does not undergo mutations to remove our construct. From another perspective, Dr. Glynn suggested that it may be difficult to take the N-terminus signal and attach it to the C-terminus. Additionally, overexpressing mitochondrial proteins in bacteria is almost impossible due to the fact that the lipid composition of the inner membrane is different from that of the plasma membrane, having a signal sequence followed by another short native domain followed by bR may mitigate this issue. Dr. Balázsi followed up by mentioning that mitochondria are not static. They constantly replicate at a rapid pace, fusing and splitting. Because of this, they have evolved membrane curvature to increase their surface area meaning that even if we have the right signal sequence, bR may still have a difficult time being inserted. To improve our design, Dr. Balázsi suggested an alternative method that we could follow: using proapoptotic proteins such as Bax which would depolarize the mitochondria for us. Dr. Gergen added upon that idea by explaining how a light inducible promoter could be used to provide strong expression, and would be the best way to activate Bax, since we were interested in optogenetic control anyways. With the help of our advisors, we learned that it would be best to switch our focus to Bax and inducing a Hypersensitive-like Response.
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After presenting our current approach to our advisors, Dr. Balázsi recommended that we read “Temporally Precise In-Vivo Control of Intracellular Signaling.” This paper discussed the use of optoXRs as a temporally precise way to control intracellular signaling pathways. This paper also helped us understand the difference between tonic and pulsatile modulation, and their impact on different physiological and pathological processes in a cell. Dr. Balázsi suggested that it would be beneficial to determine the effects of our system in basal and residual conditions. Basal conditions would denote the activity of our system with exposure to no light. Residual conditions, on the other hand, would denote the activity of our system after a certain period of light exposure. Because we cannot do this in a wet lab setting, we will likely work to model these activities.
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Since we want to integrate our project in the agriculture sector, it is very important for us to talk to professionals in this field. We reached out to HeartBeet Farms, a local organic farm on Long Island and spoke to the Co-Founder, Jennifer Ross. Jen spoke to our 2019 team about their work on Tobacco Mosaic Virus (TMV), thus she was very familiar with iGEM and our team. During our meeting, she was very open to our questions and offered us her insightful opinion on our work. When we asked her about indoor farms, Jen said that she doesn't mind that the agriculture sector is advancing. She pointed out that as a consumer and a human being, she cares more about how nutritious food is, rather than where it is grown. Our team had never thought about that. Jen Inspired us to ask indoor farms about the nutrition of their food. Another thing that Jen pointed out to us is that the reason she does not use GMOs is not necessarily because she is anti-GMO but rather that she does not have enough of the truth about GMOs. Our team felt that she was right, a lack of education does exist when it comes to GMOs. This encouraged us to think about ways we can easily teach the public about GMOs.
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A big portion of our project is dry lab, so we asked Dr. Balázsi for some prospective modeling approaches we should look into. Looking at our overview, Dr. Balázsi reiterated that we look into basal and residual conditions but in a more detailed light. With basal conditions, we could model the constant rate of RNA production in the dark to check for the dark activity of the proteins we are interested in. We also were taught that in a repressor system, there would be less leakage as we add more sites while in an activator system, there would be no way to control leakage. Another approach we would benefit from would be mitochondrial modeling. Here, we would be able to use ODEs to tell us about Bax levels over time as a function of light or promoter properties and then use cellular automata to tell us the effects of those Bax levels on the mitochondria. With these in mind, we decided that our design would be best supplemented with the following modeling approaches: nuclear shuttling of BphP1 from cytosol to nucleus in darkness and then light, threshold Bax oligomerization -- the lowest concentration of bax oligomers that guarantee cell death-- and ion diffusion (i.e. determining the effects on the levels of cytochrome c and it’s activation of the ROS pathway).
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To ensure that we got feedback on our project from various people, one of our members, Julia reached out to her Environmental Sociology Professor. In her Environmental Sociology class, Julia remembered talking about one of the biggest GMOs companies, Monsanto so she wondered how her professor would react to our work. When we talked to Dr. Shorette about our work, she was amazed at what we wanted to accomplish. When we asked her why people are so afraid of GMOs, she told us that the term has a very negative connotation. People are afraid that GMOs are bad partly due to Monsanto’s agenda and how the company has become so notorious over the years. Just like Jen, Dr. Shorette told us that people avoid GMOs because they don’t know much of the truth behind them. We had planned to tackle this challenge by creating a handbook on GMOs. When we asked Dr. Shorette what she thought of our idea, she reminded us that the general public is not likely to read a handbook. If we want to be effective in reaching the people, we should look into creating a visual that could easily teach about GMOs. After talking to her, we decided that we would not create a handbook about GMOs, but rather look into how we can educate people through visuals.
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Since we wanted to use the Bax gene as a means of inducing cell death in our plants, we read one of Dr. Hughes’ papers titled “Optogenetic Apoptosis: Light-Triggered Cell Death.” Dr. Hughes’ paper played an integral role in improving our understanding of how Bax recruitment to the other outer mitochondrial membrane (OMM) and subsequent oligomerization may be initiated with an optogenetic system. Furthermore, his work on Cry2 and Cib fusion proteins gave us a lot of inspiration as to how we may control Bax localization. When we met with Dr. Hughes to discuss his paper, he was very enthusiastic to help further our work. This meeting was extremely helpful in clearing up any questions we had with the system discussed in the paper. Additionally, Dr. Hughes gave us advice on how to introduce a proof of concept for our project, and suggested that we use an organism like yeast. Fortunately, we were also able to discuss our system and possible ways to establish a protocol for our project in terms of what the wet lab portion would have looked like. After our meeting with Dr. Hughes, we were able to start thinking about how we want to assess and measure apoptosis. He was able to inspire multiple ideas, such as we could possibly measure the release of mitochondrial contents (cytochrome c/ Smac 1/ DIABLO), DNA fragments, cleaved caspase antibodies, and the appearance/formation of vacuole.
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In hopes of understanding the role of the BphP1-PpsR2/Q-PAS1 system in more detail, we sought to reach out to Dr. Vlad Verkhusha. His work in the BphP1 system was outlined in many of the papers we have read, including “An optogenetic system based on bacterial phytochrome controllable with near-infrared light.” Our meeting with Dr. Verkhusha provided us with extremely important takeaways that significantly changed our project. Firstly, an issue with introducing the BphP1 system into eukaryotic systems (like plants) was the limited concentration of biliverdin (BV). BV is a short-lived intermediate in heme catabolism, with its apo form absolutely necessary for BphP1 to bind to PpsR2/Q-PAS1. This also changed our construct a bit, as we learned that overexpressing BphP1 (especially with a promoter like 35S CaMV) will lead to insufficient levels of BV, rending our system useless. To solve this problem, Dr. Verkhusha advised us to introduce exogenous biliverdin in cell cultures and generate a stable line of cells that express BphP1. Another inquiry we had about the BphP1 system was the trace amounts of light above 750 nm. If our system is implemented in indoor farming, the blue-red LEDs may unintentionally activate our system. This was alarming to us because we know that BphP1 in the Pfr (inactive) state is more light sensitive than than the Pr (active) state, as kinetics favors the binding of BphP1 with PpsR2/Q-PAS1. Dr. Verkhusha proposed that we use band pass filters that prevent any light above a certain concentration to pass, but, in a real-world application like indoor-farms, this solution is not feasible. Following the meeting, we turned to other optogenetic systems, namely PhyA/FHY1 & PhyA/FHL and PhyB/PIF3 & PhyB/PIF6. With this system, biliverdin availability was not an issue since they used phycocyanobilin (PCB) as their chromophore, a plentiful end product of heme degradation. Additionally, we would not have to deal with problems with the apo form unintentionally binding to its binding partner and causing dark activity. Also, this system is known to be found in Arabidopsis thaliana. One big drawback, however, is that this system is activated under 660 nm of light. This left us with two options: solving the many problems present with the BphP1 system or figuring out a way to manipulate the PhyB system. Both options, unfortunately, were extremely difficult and lengthy. Meeting with Dr. Verkhusha enabled us to see that we needed to make big changes to our design, which we set out to do in the coming weeks.
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Since our project involves manipulating A. thaliana, we decided that reaching out to a plant biologist would be extremely helpful. Founder and President of the Grow More Foundation, Dr. Creasey became a very useful asset to our team and gave us advice that significantly changed our project. Firstly, we discussed whether our BphP1 system would actually lead to the end goal we wanted: cell death. Upon reading our overview, Dr. Creasey advised us that in order to achieve our end goal, we would have to issue a more targeted approach. We discussed the idea of pluripotency, and how plants have evolved through herbivores. For example, if a small part of the plant was ingested, the plant would still survive because of the presence of stem cells. So, if we were to target just the leaves, we would not be able to kill the entire plant. She advised us that we should focus on exposing a particular light pathway from the leaves that would induce the expression of a gene target that is specifically needed for plant stem cells: RAM, SAM, and FAM. These cells are responsible for making new plant material (i.e. leaves, roots, flowers), so targeting one of these would guarantee killing the plant. Another thing we were told to look out for is, when a particular plant is subjected to stress, it rapidly advances to its flowering stage in order to reproduce plant seed before it dies. This is another reason targeting the stem cells would give us the optimal results. Additionally, we discussed possible gene targets, such as CALVATA. We were interested in CLAVATA due to the amount of literature present and its function as one of the main promoters for the development of the SAM. If we would want to continue having our system issue a hypersentive-like response, we would need two constructs: an inducible construct which would initiate expression once light hits and then another to target the ROS or immune response. One problem Dr. Creasey did foresee with this approach was that, because of its importance, CLAVATA is very well protected in the plant, and we would have to dissect quite a bit to have our system work. Because of this, Dr. Creasey proposed we look into a more elegant, efficient approach: have our light inducible system lead to the expression of a small RNA molecule. This molecule would then be allowed to transport through the plasmodesmata to the SAM, bind and silence the production of the CLAVATA gene product, and prevent the maintenance of the SAM stem cell population. In light of the problems we were facing with our current BphP1-Q-PAS1 system and Dr. Creasey’s advice, we decided to settle on the UVR8-COP1 system. Despite its poor tissue penetration, we can utilize its control over transcription to produce interfering RNAs to silence the WUSCHEL (WUS) gene. Essentially, CLV3 keeps the stem cell population small, and WUS helps it to grow, with the two acting in opposition to maintain a steady population of stem cells. We believe that targeting WUS with this new system would allow us to interfere with its transcription and translation, and cause the stem cell population to disappear through forced differentiation.
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As we would want our project to be used in indoor (vertical) farms, from the beginning of our project, we have been reaching out to vertical farms to ask for their opinion on our work. When we told David about our project, he said that he can’t see it being bad and that it would be an added benefit for hydroponics. Since this was our first time talking to an indoor farm, we asked a lot of questions about how indoor farming works. We were amazed to learn that indoor farming does not require you to have special expertise in the field. In fact, a lot of people that work in indoor farms do so because of their interests. We were sure to ask David about the nutritional value of the food grown in indoor farms. He told us that according to what he has seen, there is not much of a difference in nutritional value between food grown in indoor farms and outdoor farms. He pointed out that indoor grown food can get higher levels of vitamins as they are provided for the plants when they are being grown. Lastly, we asked David if our project would ever be implemented in indoor farms. David commented that though our project is extremely innovative, the use of GMOs in indoor farms, especially in Babylon-Micro Farms, could only be made possible when the general public begins to understand GMOs and their benefits. David helped us realize that a lot of farms make the decision to be non-GMO due to the fact that the general public is not in support of it.
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After our first meeting with Dr. Creasey, she reached out to us again and requested that we come up with detailed protocols and a tentative schedule to what we hoped to accomplish if we did have access to a wet lab. She believed that establishing these protocols and displaying them on our Wiki would promote transparency between us and the general public. If the general public was taught exactly what happens with our system and how it’s implemented, people may be more comfortable with its usage. After presenting our protocols and timeline to Dr. Creasey, we were able to improve our project to an even greater extent. Firstly, we were interested in the following transformation protocol: transformation in E. coli, then A. tumefaciens, and finally A. tumefaciens mediated transformation in the whole plant through the floral dip method. Dr. Creasey advised us that this method would be unachievable given our time frame because floral dip usually takes up to a year to work. In order to combat this, we were told to look into transient transformation. Compared to the stable transformation method we were thinking about before, transient transformation would take only 3-4 days, which is a much more feasible time frame. Here we would grow our Arabidopsis plants and perform agroinfiltration using a solution of agrobacterium containing our construct. We were also told that we should look into N. benthamiana , since agroinfiltration is mostly used in this organism. Secondly, since our new approach involves our system being exposed to UVB light, Dr. Creasey and her student, Matthew Venezia, suggested we look into implementing our system in greenhouses as well. Greenhouses are able to protect the plants against all forms of UV light so if a plant with our system implemented in it were to escape, then the plant would cease to grow. This would be especially beneficial to the environment because now, our system will be able to control a wider variety of crops. Following the meeting, we began looking into transient transformation in Arabidopsis. As it turns out, since Arabidopsis is commonly used as a model organism in plant science, researchers have established certain ecotypes (e.g. Col-0 and NahG) that would allow us to perform transient transformation. However, these ecotypes have weaker immune responses, leading them to be more susceptible to other pathogens. Additionally, although transient transformation is possible with these ecotypes, it is not necessarily efficient. N. benthamiana is widely used for transient transformation and is more efficient than transformation in A. thaliana. In light of the drawbacks of performing Agrobacterium mediated transient transformation in Arabidopsis, we decided to change our model organism from A. thaliana to N. benthamiana to establish a stronger proof of concept.
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When we reached out to Square Roots Farm for their insight on our work, we learned that a Stony Brook alumni, Maxwell, worked at the farm. Despite his busy schedule, Maxwell was very delighted to meet with us and help us in any way possible. He explained that Square Roots is very transparent because the farm understands that it is vital for people to know what they are eating. In fact, the farm really values customer feedback as “there is no end to optimation” in indoor farming. Thus, Square Roots is always looking for ways to improve their operations so that they can best serve the needs of their community. During our meeting, Maxwell highlighted a lot of the benefits of growing indoors. Such as, in vertical farms everything is controlled. This allows plants to grow in an environment where they are given exactly what they need. Maxwell mentioned that even the light given to a plant is based on the specific needs of the plant. Another benefit of vertical farming Maxwell told us about is that vertical farms, like Square Roots, only sell locally. This enables there to be extremely little time between harvest and consumption, increasing the nutritional value of indoor grown food. When we asked Maxwell about the implementation of our work in vertical farming, he brought up the point that David did. Even though GMOs may have their benefits, people see the word as an ambiguous negatively umbrella term. There is a vast lack of knowledge when it comes to GMOs and until we can deliver this knowledge properly, GMOs are not likely to have a place in vertical farming.
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Josemary Medrano from MolecularCloud/GenScript kindly invited us to a US virtual meetup for participating teams in the MolecularCloud sponsorship. Members of our team were delighted to reflect on our iGEM experience so far with Josemary and the University of Rochester (UR) iGEM team. During the meetup, each team discussed our projects, challenges this year, and where we see our work in the future. As we were discussing how this year was completely different from what we had expected, we realized all the good things that came from doing research work while in quarantine. Our team members Julia and Alexis mentioned that the loss of wet lab access this summer enabled us to focus heavily on our design. Since our research subteam spent most of their time this summer reading articles and working on the design, they were able to realize that our initial proposed design with BphP1 would not work. With the help of our advisors, we were able to refine our project so that the design would certainly work. Another great point that was brought up during the meeting was how our team was able to use Discord in order to create structure for our team. Since we were unable to meet up in person, we needed a proficient way of communication so that everyone on the team would be aware of what was happening. We were able to achieve this through Discord and it’s division of channels. Furthermore, we discussed how having multiple subteams as well as subteam leaders helped us create a better structure and order to our work. Lastly, when we were discussing where we see our project in 5 years, Gabe from RU brought up a great point. Gabe pointed out that unless someone notices our projects and continues the work, our research is not likely to survive 5 years from now. The thing that will survive, though is the education and public engagement we provide to our community. Gabe made an excellent point, stressing that one of the best ways to leave a legacy of our work is by educating the public on the topic(s) we did our research on.
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To make sure that our project could be used in more indoor farms such as greenhouses, we reached out to the greenhouse curator at Stony Brook University, Mike Axelrod. Mike has been working at the greenhouse for over twenty years and he was able to answer our questions with much detail. We learned that the greenhouse has 8 plant growth chambers that allow for temperature and lighting (both intensity and photoperiod) control. Much like in vertical farms, to control temperature and light, computerized control systems are used. Mike pointed out that temperature control is essential to ensure that plants are grown properly. When we asked Mike what were the advantages of using greenhouses compared to typical outdoor farms, he mentioned that greenhouses allow year around growth, whereas outdoor is only seasonal. Hence, crops can be grown in greenhouses even in the winter, which is not possible in outdoor farming. We were curious if the greenhouse ever grew transgenic crops. Mike told us that transgenic crops are sometimes grown in the greenhouse, but only in the growth chambers. Lastly, we asked if our optogenetic system could ever be useful for Mike’s work. He told us that although our system could be potentially used in his work, convincing the public would become the biggest challenge. Much like everyone we have talked to, Mike stressed the importance of the public’s approval when it comes to the use of GMOs.
Stony Brook iGEM 2020
