Team:Rice/Human Practices
Motivation
Our project, Thermoplant, stemmed from an idea introduced during our student-led iGEM College Course here. With rising global temperatures having an enormous effect on the world’s crop yields, we decided that we wanted to engineer a way to help plants grow better at high temperatures. However, we were aware of the common public perception that GMOs have harmful effects on people and food. Therefore, we decided that using a system involving root bacteria would be an equally effective, less invasive method to improve crop yield, while also being considerate of our community’s concerns.
Farmers' Concerns
We began by thinking of ways in which we might improve crop yields using synthetic biology. Our work began with talking to local farmers. These farmers spoke about issues they had due to climate change but were also concerned about their crops being labeled GMO in order to counteract its effects. This led us to considering how we might engineer rhizobacteria to confer greater adaptive responses. Our literature review provided us with several mechanisms by which we might improve crop adaptive response to temperature change. However, we wanted these mechanisms to only be expressed under relevant conditions.
As our project’s end goal would be most directly applicable to farmers, we wanted to collect and address their concerns throughout our project - especially at the outset. We asked a standard set of questions to each farmer we interviewed to have consistency in the structures of our conversations. We knew it was still important to collect and address local farmers’ opinions even though on paper our minimally invasive solution to climate resilient seemed like an appealing option. The questions that we asked including questions concerning the biggest problems that they encountered in the field, any particular weather phenomenon that they were having to change farming techniques for, their opinions on GMOs, if they would use our product and more. They brought up concerns such as would our bacteria wash away during heavy flooding, would we somehow in the end be consuming genetically engineered bacteria, and health impacts of GMOs in our food.
Expert Engagement
This ultimately led to conversations with an RNA biologist who taught us about temperature responsive RNA-thermometers. Using computational design we developed a set of novel thermometers which would control expression of our system. Additionally, we set out to carry out plant experiments to test the effects of the different enzymes that we were creating. By talking to an Arabidopsis expert, we learned the best procedures for plant testing. We also talked to a soil chemistry expert to learn more about the impact that our enzymes would have in the rhizosphere.
Since our project revolved around finding a way to increase crop yields amidst rising temperatures, we knew that having a temperature sensor was a must. Our background research confirmed that RNA thermometers were a possible avenue for this thermosensor, so we talked to Dr. James Chappell of Rice University - an expert in RNA STARS and other RNA folding interactions. He helped us begin to use the NUPACK design software that ultimately influenced our RNA thermometer design, as well as provided a great amount of advice and reference papers.
While the goal of our project was to create an engineered rhizobacteria that would increase crop yields, it wasn’t yet feasible to test our rhizobacteria amongst actual crops. Thus, we planned to use the model plant organism Arabidopsis thaliana. We met with Dr. Bonnie Bartel of Rice University’s Biosciences department, who works extensively with Arabidopsis thaliana. We were able to learn beyond standard protocols and learn techniques such as vertical plating. Even after our initial meetings with Dr. Bartel and graduate students, we were able to continue our plant experiments with the lab’s constant support.
We chose our three component enzymes IAA, ACC deaminase, trehalose synthase for their abilities to make the plant more drought and heat resistant. These enzymes were also naturally found in Pseudomonas putida, so we did not have to worry about introducing new pathways and instead could focus on upregulation. While we knew the effects of the enzyme products in the plant, we were interested to see if there were other effects in the environment. We talked to Dr. Masiello, a soil chemistry expert, who helped us to understand more of the mechanics behind the different enzyme products. We also discussed the composition of soil and different soil environments our bacteria would need to live in. Overall, she thought that our enzyme products had potential to increase water retention and she helped to justify our enzyme selection.
Environmental Impacts
After the design portion of our project we began to think about our work more broadly, namely how it would affect the local environment. We spoke to a local environmental advocacy group who helped us develop an impact report outlining the potential ramifications of our project on the environment and strategies for mitigating any undesirable side effects.
Impact Report
While talking to stakeholders, we realized that many of the concerns stemmed from misconceptions in the foundation of our project. Temperatures are rising, and while the decreases in crop yield are not overtly obvious -- the global decreases add up to a dangerous problem. Additionally, while there are some controversies surrounding GMOs, our project provides a safe option that has the potential to make a large impact. We decided to collect these reasonings and flesh them out in detail in our impact report which can be accessed here. We discuss the global need for our project and address concerns that have come up with in our conversations with various stakeholders.
Environment Texas is a local environmental organization that targets different environmental problems in our local community. We met with Jen Schmerling, the deputy director in Houston for Environment Texa, to learn more about the relevance of our project in our local community. We discussed how our project is needed in our community because it is the intermediate that can help us maintain crop yields while temperatures rise. She emphasized the importance of projects like ours that dealt with the current effects of rising temperatures, because our society needs both long term solutions and current solutions. Miss Schmerling also brought up points of concern with our project that we could strengthen to make our product more usable and safe. Environment Texas is not currently targeting falling crop yields but they were willing to work with us to help make our Impact Report a resource for our community.
Bioinvaders is a local startup that works to remove invasive species out of compromised environments and turn them into educational resources. Our team wanted to learn more about what sort of problems arose when non-native species were introduced to an ecosystem. Population control is a large problem that has an enormous economic and environmental impacts. Bioinvaders deals with larger invasive species and turns them into educational experiences. Containment and controlling populations are difficult matters but it is imperative that our bacteria can be contained within the intended area. We do not want to aid invasive plants take over an ecosystem by boosting their growth on accident.
Diversification of Strain
After seeking answers to these questions, we concluded that while Pseudomonas putida is a fairly versatile root bacteria, it is not a perfect, all-encompassing solution. However, when we were initially choosing our model organism, we were also limited by which rhizobacteria we could actually access for testing, prompting us to choose P. putida. Thus, we propose 2 other strains of rhizobacteria as options for diversifying our genetic circuit to ultimately create 3 strong candidates for farmers to use:
Azospirillum brasilense is a good candidate. It already produces or has been modified to produce all of our enzymes, is one of the more well-characterized plant growth-promoting bacteria species, and it's versatile in which crops it'll work with.
The other strain that we chose as our last candidate is Bacillus amyloliquefaciens QST713 because it is another well researched rhizobacteria. It has a different advantage as it has been shown to have antifungal activity, which could a problem that farmers might have to account for. By having three potential chassis for our construct, we could theoretically have a more applicable product that is more useful to farmers.
Biosecurity
Furthermore, we participated in a biosecurity workshop where we investigated different ways our work could be used for malicious purposes and steps we could take to avoid that possibility. This led to the development of additional modules that provide biocontainment to the engineered microbes.
As our proposed end product would involve farmers as our primary stakeholders, we prioritized communicating our project proposals to them to get their input at the outset of our project. After discussing our project with local farmers, we decided that the easiest way for them to implement it would be a product that they can apply to their soil. However, this method of introduction to plants has a significant risk of contamination outside our system. We needed to ensure our system would not pose a risk to the outside environment.
After attending a biosecurity training seminar led by two graduate students, we realized that we needed a way to ensure that our engineered bacteria could not escape into the outside environment. Ronchel et al (1998) designed a kill switch shown to work for soil rhizobacteria that induces cell death in the absence of the inducer, methylbenzoate. When the bacteria is added to crop roots, regular applications of methylbenzoate ensures its survival. If the bacteria escape the field, the gef gene will be induced, which ultimately kills the bacteria by interfering with the cell membrane potential. When the circuit below is combined with the Tol plasmid, it creates an effective, geographically based kill switch. Including these constructs with our own plasmids would address the biosecurity concerns raised about how we could stop our bacteria from propagating into unintended environments.
Additionally, we participated in a biosecurity workshop in which we investigated how our project’s methods could potentially be used for malicious purposes, and we discussed steps that we could take to avoid that possibility.
Real World Translation
Looking ahead we decided to speak to a patent lawyer and discussed the process and limitations of turning our project into a commercial product. While we ultimately did not move forward with this avenue it provides insight into how to translate our work and maximize the effectiveness of the resulting patent.
We talked to Melissa Schwaller, a partner at Ramey and Schwaller, LLP and registered patent attorney, about our project’s future potential. During our conversation, we encountered tough questions and were forced to think about the novelty of our project. We discussed the disadvantages and advantages of publishing in a scientific paper versus filing for a patent. The novel aspect of our project is our RNA thermometer sequences, even though they were created using an algorithm based off of a software. In addition, we discussed the intellectual property and how it was dependent on one’s job. While it was helpful to learn about the potential future of our project, time limitations did not allow us to pursue this path further.
iGEM Project Selection Analysis
Following all of the steps we took above, we were curious about how other teams dealt with these challenges, particularly after a Skype conversation with the Oxford team revealed that few European teams pursue agricultural projects like ours because of the stringent regulations on GMOs there. We invited different teams around the world to complete our surveys about factors influencing their choice-of-project. We believe that factors glimpsed from these surveys could help future teams select projects with maximal benefit to their local communities. To see our complete meta-analysis, Click here
Conclusion
In conclusion our project was developed to address an emerging need not only in our community but globally. We investigated how our work might best serve those who will ultimately rely on it, how to best ensure we do the least harm and the most good, and how to translate our work to the field.
References
Ronchel et al. (1998). Characterization of Cell Lysis in Pseudomonas putida Induced upon Expression of Heterologous Killing Genes. Appl. Environ. Microbiol 64 (12) 4904-4911.