Team:UMaryland

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An Apeeling Solution
to Panama Disease

Projects

Saving the Cavendish banana

Outreach

Increasing access to synthetic biology for high schools

Parts
Modeling
Notebook

About Us

Learn more about UMaryland iGEM

← Projects

From saving the Cavendish to expanding synthetic biology

An Apeeling Solution

to save the Cavendish

Cas9 Mutant Screening

Screening for mutants using CRISPR/Cas9

Lab-in-a-box

Low cost DIY lab equipment

Metal Detection

Teaching synthetic biology using real world examples

← Outreach

Reaching out to the community about synthetic biology

Knowing Panama Disease

Feedback, concerns, and feasiblity

Teaching the Next Generation

of synthetic biologists

Talking with the Community

about synthetic biology

← About Us

UMaryland iGEM - Since 2014

Students

The next generation of scientists

Advisors

Guiding our efforts

Funding

Providing support for the team

Acknowl-
edgements

Those who helped us get here

← Application

Contributing to the scientific community

Parts

Contributions to the Registry

Modeling

Applying engineering principles

Collabor-
ations

Working together with other synthetic biologists

Notebook

Follow our progress throughout our experience

An Apeeling Solution to Panama Disease

The Issue

The banana is the most popular fruit in the world, with a whopping 160 million tons produced annually. In America, we think of the banana as a fruit, something we love but could live without. However, the same cannot be said for over 400 million people in developing countries. These individuals rely on bananas as their biggest, and sometimes, sole source of calories and nutrition. Terrifyingly enough, 7% of the world’s population could lose its sole source of food as the banana crop is being ravaged by a devastating pandemic: Panama disease.

Panama disease is caused by the fungus Fusarium oxysporum f. sp. Cubense race 4, which is deadly to the Cavendish banana. This fungus is able to first infect the roots of the plant, secreting different hydrolytic enzymes, and subsequently infect the xylem, blocking water and nutrient flow, leading to the death of the plant. Symptoms, however, are typically not visible until four months after infection, providing ample time for the fungus to spread without notice. Additionally, the fungus remains in the soil in the form of spores for up to a decade, ready to infect the next set of plants. Therefore, there is a need to develop a method of protecting the Cavendish banana from Fusarium oxysporum and eliminating the fungus from the soil.

Our Approach

Many eukaryotes produce a class of proteins known as thaumatin-like proteins (TLPs) in response to attack and stress. Some of these TLPs are known to have antifungal properties against F. oxysporum. Among these are the TLP from Oryza sativa (rice) and Arachis diogoi (wild peanut plant).

Current methods of fungal inhibition are too detrimental and cannot be used for extended periods of time. However, recent studies have shown that biocontrol methods using disease-suppressive soils have been quite effective at fungal inhibition. Bacillus amyloliquefaciens (BA) NJN-6 is a common Bacillus strain found in the Cavendish banana rhizosphere. BA has been found to produce some antifungal compounds that are fairly effective at suppressing fungal growth. By combining BA with compost to make a fertilizer and applying it to soil already infected by F. oxysporum, the susceptibility of the banana plants to the fungus was greatly reduced. While quite effective, the bacteria alone is unable to kill the fungus.

As a result, we propose to transform BA to produce an antifungal TLP. Then we will again combine this with compost to create a fertilizer that is even more potent. Since TLPs are known to actually be able to kill the fungus in addition to being able to inhibit spore germination, this transformed BA has the potential to be more effective against F. oxysporum.

Why Choose Our Approach

There is currently a lot of stigma around and distrust of genetically modified foods. As a result, we wanted to focus on creating a genetically modified organism that would serve as a probiotic treatment for banana plants.

Our Design

A.Bacillus amyloliquefaciens, a bacterium that grows synergistically with the roots of the Cavendish banana plant, senses Fusarium oxysporum, the fungus causing Panama disease. B. In response, B. amyloliquefaciens produces TLP, an antifungal, to fight off the pathogen.

Summary of components

There is currently a lot of stigma around and distrust of genetically modified foods. As a result, we wanted to focus on creating a genetically modified organism that would serve as a probiotic treatment for banana plants.

Our initial steps will involve making plasmid designs for E. coli (as E. coli will the the initial chassis used for proof of concept). Our E. coli must be able to sense F. oxysporum. Upon presence, the bacteria should produce the antifungal TLP and there should be a mechanism for the TLP to be able to interact with the fungus. In addition, the E. coli must also be able to survive in the conditions that result from the presence of the fungus. In summary, the basic components of our circuit are a F. oxysporum sensing system, a fungal killing mechanism, and a chassis survival mechanism.

We wanted to find a way to uniquely sense F. oxysporum, such that a response is only triggered in its presence. We found that F. oxysporum produces fusaric acid, a small weakly acidic (pKa 1.1) mycotoxin and phytotoxin that is able to diffuse across the cellular membrane. Fusaric acid contributes to the virulence of the fungus. It causes super polarization, suppressed H⁺ pumping, and K⁺ leakage in plant cells. It also reduces oxygen absorption by mitochondria and causes malic acid oxidation. Downstream effects include the creation of reactive oxygen species and changes in membrane polarity and permeability . Fusaric acid is also toxic to several bacterial species, including E. coli, posing a possible issue for testing that we addressed later on.

Different bacterial species have shown unique sensitivities to fusaric acid. Some organisms that are resistant to the compound are able to produce efflux pumps in its presence. These include Pseudomonas putida and Stenotrophomonas maltophilia, which both incorporate an efflux system that is repressed when there is an absence of fusaric acid. Our initial plans were to design a plasmid with TLP regulated by a promoter that was repressed by these fusaric acid-sensitive repressors. However, the operators have not yet been identified, making it difficult to adjust the system to E. coli.

Fusaric acid has also been shown to suppress the production of an antimicrobial compound, 2,4-diacetylphloroglucinol (DAPG), produced in Pseudomonas fluorescens. In this system, fusaric acid is able to induce the phlF repressor that is then able to suppress expression of the adjacent phlA gene. Concentrations of 500uM completely repress transcription, 300 uM produce some inhibition, and 100 uM have no effect. In 2015 Glasgow combined the characterized operator of the phlF sensitive promoter native to P. fluorescens and incorporated it into a strong E. coli Anderson promoter. We will use both the repressor and synthetic promoter in our design to make our bacteria sensitive to fusaric acid. The DNA following this part will have suppressed transcription in the presence of fusaric acid.

The natural system follows the sensing of F. oxysporum with inhibition of the next component, which was opposite of the circuit we wanted. Therefore, we created an intermediate step in the circuit that turned inhibition into activation by having the phlF repressor act on a gene responsible for repressing the killing mechanism in normal conditions.

The killing mechanism required us to produce an antifungal that will target F. oxysporum. For this part, we chose to incorporate rice thaumatin-like protein (TLP) into our design. Plant TLPs are defense pathogenesis related proteins of family 5. Rice TLP has shown antifungal activity both when over-expressed in the rice plant and also when expressed in E. coli. The exact mechanism is still being studied, but TLPs are suspected to disrupt the cell walls of fungi by binding to glucans and acting as glucanases. β-glucan is the most abundant polysaccharide found in fungal cell walls and its degradation destabilizes the fungal cell wall. TLP may also provide resistance to fungal disease as a xylanase inhibitor, inhibiting an enzyme that breaks down a component of plant cell walls. Because literature sources have confirmed the antifungal properties of rice TLP, as well as its ability to be produced by E. coli, we chose to use this protein as our fungal attack piece.

Amounts of 5 ug of rice TLP extracted from recombinant E. coli were enough to inhibit fungal growth of 7 mm mycelial discs and concentrations of 500 ug / mL of TLP showed over eighty percent inhibition of spore germination of F. oxysporum f. sp cubense.

From the components we had gathered through research, we created three initial design ideas.

TetR Design. In this design, the TLP gene is under a pTet repressor and is therefore repressed in the presence of TetR. TetR expression is regulated by the phlF repressor, which is induced by fusaric acid. When fusaric acid is not present, phlF is not able to repress and TetR is freely transcribed and translated, therefore binding to pTet and repressing TLP transcription. In the presence of fusaric acid, phlF is activated, TetR is no longer transcribed and degraded in the cell, thereby allowing for the transcription and translation of TLP.

Antisense Design. In this design, translation of TLP is inhibited by the expression of antisense RNA. Similar to the mechanism of the TetR design, TetR is replaced by TLP complementary DNA.

T7 Design. This design is similar to (A), except a T7 polymerase is controlled by pTet, which is repressed by TetR when fusaric acid is not present. The TLP is transcribed by this polymerase. Only when the T7 polymerase is expressed can the TLP be expressed.

Design	Pros	Cons
TetR	Simple, easiest to construct	Low expression of TLP (weaker promoter), iffy time scale (would TetR be degraded fast enough)
Antisense	Pretty neat idea, more novel, quick RNA degradation rates, smaller plasmid	Need correct ratio - causes need to limit TLP transcript number, slightly harder to make
T7	Optimized for production - T7 very fast, efficient polymerase	Higher metabolic toll, bigger plasmid
All		Unsure if E. coli will survive before production of TLP Unsure if TLP will be able to go outside of cells Unsure if amount TLP produced will show significant changes in fungal growth

All three of these designs have the same logical format: If there is no fusaric acid in the environment, something will inhibit TLP production. When fusaric acid is present, that inhibition will be taken off and expression will occur.

After some mathematical modelling and assessments, we decided that the antisense system was the best option, and so it was incorporated into our final design.

Additionally, we initially predicted that eventually the overexpression of the TLP will kill the cell causing TLP release to the exterior surroundings. This TLP would be available to interact with and attack the fungi. However, we were advised that this method was not reliable due to the possibility of protein aggregation and so we decided to secrete the protein instead. We found several iGEM teams had worked with protein secretion in E. coli, including Unicamp - EMSE Brazil 2011 and used the information they provided as well as their results to create a secretion part. We found that gram negative bacteria, like E. coli, have a secretion system that involves HlyB, HlyD, and TolC proteins. HlyB is an ATP-binding cassette transporter. HlyD is a membrane fusion protein, and TolC is an outer membrane protein. HlyB and HlyD are not present in all strains, so there was a necessity to express them in our bacteria and incorporate the two proteins into our part. In order for a protein of interest to be secreted through this system, it must be labeled with an HlyA signal sequence, which we decided to add to the C terminus of our TLP.

We integrated all the different parts and components to one design.

Overview of circuit design. The phlF repressor and TLP genes are always constitutively expressed. A. Without Fusaric Acid: Without fusaric acid the repressor remains “off.” An antisense RNA that compliments the TLP gene is expressed under the pPhlf promoter. TLP translation is inhibited by the binding of TLP RNA to the antisense RNA. B. With Fusaric Acid: The phlF repressor activated by fusaric acid. Once activated, phlF is able to bind to the operator on the Pphlf promoter, thus inhibiting the expression of the antisense RNA. Without the antisense RNA inhibiting translation, TLP is produced. C. Protein Secretion: The TLP is labeled with an HlyA signal sequence that will allow it to be secreted out of the cell via an HlyB-HlyD-TolC transport complex.

Plasmid Design. Our final plasmid for an E. coli chassis includes a “sensing” system controlled by the phlF repressor and the PphlF promoter it acts on, a “silencing” mechanism with antisense TLP expressed in normal conditions, a “killing” system that produces antifungal TLP, and an export system that utilizes the HlyA signal and HlyB and HlyD export proteins.

Cas9 Mutagenesis Projects

Our Design

We are attempting to apply Cas9 in a novel way by using it to screen for mutants. If we introduce Cas9 with an sgRNA identical to a region of interest within a gene, it is possible to cleave plasmids containing an unmutated version of that gene. Coupling this method of selection with random mutagenesis creates a powerful tool that can greatly reduce the amount of screening that needs to be completed following a mutagenesis operation. This screening method creates a way to discover the functions of specific regions in genes, or as a tool for directed evolution of a protein.

Understanding Panama Disease

iGEM projects reside in their context, and the purpose of human practices is to learn more about the problem and the situation that surrounds the problem. This requires a multi-disciplinary approach that involves discussing our project with stakeholders that are from a variety of backgrounds. To understand the potential impact of our project, we contacted multiple individuals in a wide variety of fields and asked for their feedback and their possible concerns.

Business / Administration

From the business perspective, we contacted Beyond the Peel, an organization that aims to empower independent banana farmers in Ecuador and Peru, and to import fair trade organic bananas. We were told that the lack of Panama disease in South America meant that farmers are more concerned about local diseases that are currently afflicting the banana plant more than a disease localized in Asia and Africa. These diseases, along with the daily concerns of the weather and pests, are the farmer’s primary concerns, although we were told that if we had a good product that would protect the farmers, and if the disease has begun to manifest in the Americas, there would be no backlash against using foreign or GMO solutions.

In addition, we learned that there are many reasons beyond the technical challenges why the Cavendish banana is the major crop being imported. This mainly is due to infrastructure challenges, since shipping times, routes, and distribution mechanisms all depend on the Cavendish, whose ripening properties are well known. In addition, consumer demand for bananas that aren’t the Cavendish is miniscule, and no investments on building the infrastructure for different types of bananas that may be more resistant to Panama Disease, isn’t being made. We also spoke to the United Nations Food and Agriculture Organization (UN FAO) about our project and received the perspective of a policy administrator. We found that there was a World Banana Forum hosted by the UN FAO that aims to tackle labor, disease, and agricultural issues through a collaboration of industry, academia, and governments. From our conversations with those not in the scientific community, we found that biosafety and biocontainment was a major concern, which included the potential pathogenicity of our genetically engineered bacteria.

Scientific

We also spoke with Dr. Juan Robalino, a researcher tackling the problem of Panama Disease, about our project, and we discussed the regulatory hurdles of conducting synthetic biology overseas. He is from Ecuador, and he founded / is the CEO of the startup Cronicas, which aims to develop crops that are more resistent to virulent and fungal infections. As a researcher in genetically modified bananas, he was able to explain that outside of the US, the precautionary principle with regards to recombinant DNA is applied to even research in the lab, requiring a permit which may take years. Since bananas are not affected in the Americas, research has to be conducted in Southeast Asia, which is both the major producer of bananas consumed outside the Americas and the country most devastated by the disease. The governments of the Southeast Asia also view this as an industry issue that companies need to solve, and that governments do not need to intervene on this issue. Currently, there are tissue banks (which are used to culture and grow the banana plants) in Taiwan that are working on finding resistant varieties. He also notified us that there isn’t much of a push towards solving this crisis because banana markets are depressed, meaning there is overproduction and a very low cost of bananas. He forecasted that once bananas become a more valuable crop, there will be a more concerted effort.

Regulatory

To understand the regulatory hurdles, if they were to be tested in the US first, we spoke with Dr. Eric Olson and Dr. Carrie McMahon of the Federal Drug Administration and Dr. Chris Wozniack of the Environmental Protection Agency, the two organizations that would regulate the use of our system. We learned that Bacillus amyloliquefaciens has been used as a fungicide in the past, and that thaumatin, which is similar to TLP, has preliminary evidence to be safe for ingestion by humans. We also learned that the FDA wouldn’t regulate our system because it does not directly impact the food and that the US Department of Agriculture would not regulate our system because we are not engineering plants, leaving only the EPA as the sole regulator. Although the regulation of genetically engineered bacteria versus a symbiont is more strictly controlled, there is enough precedence to consider our project more feasible compared to the engineering of banana plants themselves.

UMaryland iGEMers

UMaryland iGEM is made of dedicated undergraduates who are passionate about improving the world through synthetic biology. They are from a variety of backgrounds, majors, and interest that contribute their talents through lab work, outreach, fundraising, wiki coding, video editing, social media, and photography. They dedicate their time through a spring seminar, summer work, and finishing up of the project in the fall.

	Asha Kodan Asha is a rising sophomore who is majoring in Biology with a minor in Religious Studies. This is her first year on the UMaryland iGEM team! In addition to her passion for synthetic biology, she is an avid writer and opinion columnist for the Diamondback Newspaper. She also helps with behavioral inhibition research at UMD's Child Development Lab. During her free time she enjoys traveling and watching true crime documentaries.
	Cameron Harner Cameron is a senior in Bioengineering. After graduating he plans to work as a consultant in the healthcare industry. He joined iGEM to get hands on lab experience and work on a project with real world applications. Outside of lab he enjoys sports, game of thrones, and just about anything outdoors.
	Deven Appel Deven is a junior bioengineering major on the pre-medical track. With a desire to explore synthetic biology, he found iGEM had the perfect goals for him. iGEM also provided a first-hand look at how these principles of biology and engineering could be applied to actual, beneficial goals in the world, and he was happy to see such a passionate, determined team around him at the University of Maryland. Deven is also the president of an a cappella group and assists with a biophysics lab on campus.
	Chun Mun Loke
	Cynthia Uzoukwu Cynthia is a senior with a double major in bioengineering and criminal justice. After she graduates, she hopes to go on to medical school to become a neurosurgeon. She joined iGEM because she likes that students get to choose their own research topic, based on their analysis of what real life problems synthetic biology could help combat. Apart from science, she likes anything involving criminal law.
	Jacob Premo
	Joyce Song Joyce is a sophomore neurobiology & physiology major. She joined iGEM because she loved the concept of a student-ran research team that tackles real world issues with synthetic biology. In her free time, she loves to go play with her dog, go on hikes, and binge-watch Netflix.
	Paula Kleyman Paula is a junior studying bioengineering. This is her second year on the UMaryland iGEM team. She really enjoyed working on the team the year before and learning how to design and create a novel synthetic biology project and came back for more. She is also interested in pursuing a career in education, possibly finding a way to integrate her biology and engineering knowledge with classroom work. Her favorite activities outside of academics are running, singing, and playing guitar (but not at the same time).
	Richa Beher Richa is a sophomore Cell Biology and Genetics major at the University of Maryland who aspires to become a physician-scientist. She joined iGEM to gain research experience and explore synthetic biology applications with like-mided individuals. On campus, she is part of service organizations, Terrapin Trail Club, and the Club Triathlon team! In her free time, she enjoys eating good food, backpacking, listening to music, and hanging out with friends.
	Rohith Battina Rohith is a sophomore biochemistry major at UMD. He is interested in pursuing a career in the field of infectious diseases research. He enjoys doing iGEM because of its application to real world problems and how it fosters teamwork. In his spare time, he plays tennis and reads books.
	SangHo Jee He is currently coding the wiki for the team.....
	Seth Cohen Seth is a junior biochemistry major at the University of Maryland. Outside of science his favorite things to do are play basketball and write fiction. His favorite food is probably falafel on pita bread with tomatoes, cucumbers, feta, lettuce, tahini, and hummus. His favorite animal is definitely the elephant. Another kind of interesting fact about him is that he is vegetarian. That's pretty much all you need to know about Seth!
	Thea Orstein Thea Orstein is a rising Junior at the University of Maryland, College Park, majoring in Bioengineering with a concentration in Biotechnology and Therapeutics Engineering. After graduating from UMD, She plans to pursue a PhD in Biophysics or Molecular Biology. This summer, she is working as a lab member and as a project developer on the UMaryland iGEM team. In her free time she enjoys running, hiking, and hanging out with friends.
	Yuzhu Shi Yuzhu is a sophomore biochemistry and microbiology major. She loves doing research and thought that iGEM would be a great way to further pursue that interest. Her other hobbies include trying out new foods and restaurants, going to the gym, and hanging out with friends.

UMaryland iGEM Advisors

UMaryland iGEM is lead by two faculty advisers: Dr. Edward Eisenstein in the Fischell Department of Bioengineering, and Dr. Jason Kahn in the Department of Chemistry and Biochemistry. We were founded with the help of Dr. Boots Quimby, former Assistant Director of the Integrated Life Sciences Honors Program at the University of Maryland, College Park. They help evaluate project proposals, attend regular lab meetings, connect us to resources on and off campus.

	Dr. Jason Kahn Dr. Jason D. Kahn is a biophysical chemist who studies protein-nucleic acid interaction and engineering. He is best known for studies of DNA looping, bending, twisting, and cyclization, as well as hybridization thermodynamics for modified bases. He teaches a variety of chemistry, biochemistry, and molecular biology courses, which he credits for initiating his interest in synthetic biology. Dr. Kahn was a graduate student at UC Berkeley and a post-doc at Yale before coming to Maryland in 1994.
	Dr. Edward Eisenstein Dr. Edward Eisenstein is a Fellow in the Institute for Bioscience and Biotechnology Research and an Associate Professor in the Fischell Department of Bioengineering at the University of Maryland. Trained in modern structural enzymology, his current research interests are focused on protein and biosystem engineering for discovery and application in plants and microorganisms.
	Dr. Booth "Boots" Quimby Dr. Quimby is the former Associate Director of the Integrated Life Sciences honors program in the Honors College at the University of Maryland. Prior to joining the Department of Cell Biology and Molecular Genetics at UMD as a full-time instructor, she earned her Master's of Arts in teaching from the University of South Carolina, after which she taught high school science in Atlanta, Georgia for eight years. She then returned to graduate school and received her doctorate in genetics and molecular biology from Emory University.
	Naren Bhokisham Naren is a Graduate Student in Molecular and Cell Biology at the University of Maryland, College Park. He received his undergraduate degree in Industrial Biotechnology from St. Joseph’s College of Engineering, Anna University, India. He works in the intersection of synthetic biology, metabolic engineering and biomaterials, involving assembly of enzyme cascades on various interfaces to generate small molecules and engineering microbes to display novel phenotypes in response to small molecules. Apart from science, his pursuits include traveling, running and latin dancing.