Team:Lambert GA/Poster
Poster: Lambert_GA
Advisors: Mrs. Janet Standeven, Dr. Brittney Cantrell
| Shivaek Venkateswaran PPB | Sahana Narayanan Pho | Human Practices | Marketing | Video | Jenny Nah PPB | Human Practices | Aryan Singh PPB | Modeling | Edgar Robitaille PPB | Lyophilizer | Andrew Bae PPB |
| Samhitha Yeleti Human Practices | Alice Hou PPB | Modeling | Marketing | Manasvi Gupta Pho | Human Practices | Video | Richard Jiang PPB | Zoya Mir Human Practices | Marketing | Video | Monica Cho Pho | PPB |
| Regina Ooms Human Practices | Michelle Jing PPB | Marketing | Sachintha Ashok Nar | Vineeth Sendilraj Lyophilizer | Wiki | PPB | Melanie Kim Modeling | Kathy Ye Nar | Video |
| Madhav Gulati Plate Reader | Wiki | Neha Lingam Human Practices | Marketing | Aditya Prabhakar Lyophilizer | Saif Khan Nar | Human Practices | Sishnukeshav Balamurali PPB | Hari Mudigonda PPB | Video |
| Hannah Noh Human Practices | Video | Pranav Kanthala PPB | Varun Sendilraj Plate Reader | Stan Lee Lyophilizer | Dr. Brittney Cantrell | Mrs. Janet Standeven |
Integrated Human Practices
Lambert iGEM continued AgroSENSE from the previous season with a focus on making our biosensors safer and more efficient. We collaborated with stakeholders including farmers, state legislators, teachers, local libraries, and nonprofit organizations to determine AgroSENSE’s implementation in society.
Problem
Last year, while developing our nutrient biosensors, we noticed a high prevalence of root rot in our hydroponics system that were caused by plant pathogens Fusarium oxysporum and Phytophthora cryptogea. We reached out to Mr. Clint Crowe, owner of Sweetwater Urban Farms, to further understand the consequences of root rot in commercial systems and how these infections significantly reduce crop yields. From Dr. Bhabesh Dutta, we learned that root rot is highly prevalent in Georgian agriculture, and he encouraged us to continue developing riboregulated switches to detect plant pathogens at their spore stage.
In order to gain the government’s insight on the implementation of our biosensors, we consulted the Georgia Department of Agriculture, specifically Commissioner Gary Black. We were prompted to extend AgroSENSE towards soil-based farms along with hydroponics after Commissioner Black informed us that most Georgian farmers were more inclined to use traditional forms of agriculture. To address biosafety concerns farmers and other government officials brought up regarding the use of bacterial biosensors with crops, we reached out to Dr. Adam Silverman and Dr. Saad Bhamla to transition our nitrate sensors into cell-free systems and construct a frugal lyophilizer to preserve these cell-free lysates and whole-cell biosensors during transportation. The Georgia House of Representatives Chairman Todd Jones and Georgia BioEd Institute guided us through the process of drafting a regulatory proposal for our biosensor implementation.
Proposed Implementation
Lambert iGEM consulted the Georgia Department of Agriculture to learn about the implementation process of our biosensors in Georgia’s agricultural market. From our discussions with the Department Commissioner Gary Black, we learned that there are currently limited governmental barriers standardizing this process as biosensors are emerging innovations in the agricultural industry. As a team that works closely with E. coli, we know of the potential hazards bioengineered products pose to both farmers and consumers if handled incorrectly. We collaborated with Todd Jones, a member of the Georgia House of Representatives, and Ashley Haltom, the Vice President of Government Affairs at Georgia Bio, to develop a regulatory proposal to standardize the use of biosensors.
Science Communication
Cameroon
Lambert iGEM collaborated with ONASI Bilingual College (ONASI) in Ebolowa, Cameroon to provide their students with a series of synthetic biology workshops and corresponding laboratory activities that align with the Cameroon national high school standards and the Georgia Standards of Excellence for Biotechnology. Once we had a clear understanding of the curriculum, we arranged to develop a series of virtual workshops and send materials for a teacher training that would take place in early September (see Fig. 2). In the future, we anticipate Mr. Onana will be able to share the training framework with the ministry of education in Ebolowa and promote the use of a community lab kit that would provide materials to expand this opportunity to other schools in the Mvila school district.
Storybook
We wrote and illustrated a children’s storybook, Grow and Glow, targeted towards elementary schoolers to introduce them to the fundamental concepts of synthetic biology. We partnered with our local library and elementary school, Sharon Forks Public Library and Sharon Elementary School, respectively, to host storybook readings and hands-on activities. We referred to the Georgia Standards of Excellence for third, fourth, and fifth grade to ensure appropriate lexile and content in the storybook. To gauge the effectiveness of our readings, we conducted pre and post formative assessments. The responses, along with the Q&A session, demonstrated student interest and retention levels for synthetic biology.
Cloning Webinar
Lambert iGEM hosted a collaboration to teach newly developed iGEM teams about the restriction enzyme cloning workflow. This webinar introduced each step of the workflow and briefed participants on how to build an effective plasmid and insert. We asked poll questions throughout the webinar and conducted a post-webinar survey to determine how well members were able to retain the lesson. We were able to determine that the webinar had successfully taught the concepts reviewed through recorded responses that demonstrated significant understanding.
Background
A plant requires several essential nutrients, maintained at proper concentrations, to grow and function properly within a hydroponics system. From our research, we identified that inorganic phosphate levels are difficult to precisely detect and test since testing kits are often costly, time-consuming, and inaccurate. To address these issues, we developed our two-year project, AgroSENSE, centered on designing and characterizing a low-cost, highly-sensitive phosphate biosensor. Implemented with modular hardware and software, our characterization curve aims to provide an easy way for hydroponics users to accurately detect the specific phosphate concentrations within their system. We also lyophilized our biosensor cells to eliminate biosafety hazards in distributing our biosensors to the public.
Part Description
BBa_K2447000, utilizing the Pho Regulon signaling pathway native to E. coli, is an extracellular phosphate sensor with a PhoB promoter and a GFP reporter.
As shown in Figure 1, BBa_K2447000 consists of an inducible promoter (BBa_K116401) that is activated by the binding of phosphorylated PhoB transcription factor. The natural genes of the Pho Regulon signaling pathway are replaced with a GFP reporter (BBa_E0040) so the activation of the promoter following low levels of Pi would result in GFP expression.
Characterization
Through extensive research, our 2020 team discovered that phosphate concentrations between 50μM and 100μM are ideal for plant growth in hydroponics systems. Although we proceeded to test GFP expression of our biosensor on a phosphate level range of 0μM to 150μM for detailed characterization, we repeatedly saw an abnormal bump around concentrations from 120μM to 150μM.
In order to resolve this issue, we reached out to professors from the Georgia Institute of Technology and Emory University and decided to focus on a more accurate and detailed characterization only for phosphate concentrations under 100uM. Additionally, we decided to add MOPS wash steps and a vortex step in our protocol to prevent residue LB and uneven distribution of cells from affecting our data. These modifications allowed us to achieve an enhanced characterization curve (Figure 2).
Sample Testing
To measure the efficiency of our biosensors, we ran numerous tests from various water sources. We used our biosensors on samples from local hydroponics systems and lakes to determine the inorganic phosphate concentration. To double-check the efficacy of our biosensor, we ran a phosphate concentration analysis on all our samples (Figure 3). The concentration shown on the concentration analysis compared to the sample testing results from our biosensors’ characterization curve had no significant difference, which supports the claim that our biosensors are able to detect highly specific extracellular inorganic phosphate concentrations.
Nar
Before beginning experimental testing of the Nar biosensor, Lambert iGEM developed a deterministic Ordinary Differential Equation (ODE) model of the Nar pathway with part BBa_K1682018. The ODE model provides a visual representation of the signaling pathway along with stimulating GFP expression from the nitrate biosensor to measure the range of nitrate concentration detection.
The model’s predictions followed a similar trend as HKUST-RICE 2015 iGEM team’s characterization data of the part. However, the scale of GFP expression differed since our model simulated GFP expression of a single cell while HKUST-RICE measured that of an entire culture of cells.
Pathogenicity
After selecting target pathogens to detect, Lambert iGEM constructed a Susceptible-Infected-Removed (SIR) predictive model of Fusarium’s spread in hydroponics. It mainly bases itself upon the parameter, basic reproductive number, which is the average number of other healthy plants infected by one previously infected plant.
With data from Sweetwater Urban Farms and online literature, this model thus predicted the following showing the expected infected plants and deaths based on one initially infected plant in a system of three towers all holding 248 plants combined.
Overview
Nitrate is also an important nutrient for plants in hydroponics systems. Similar to the phosphate biosensor, commercial cost-efficient nitrate sensors are often imprecise and time-consuming. Therefore, as another part of AgroSENSE, we engineered a cost-efficient, user-friendly biosensor to detect nitrate using E.coli’s native nitrogen detection system: the Nar Operon. With this, in tandem with our frugal plate reader and lyophilizer, hydroponics farmers can precisely measure the nitrate concentration in the hydroponics system.
Background
We utilize the native Nar Operon signaling pathway in E.coli to detect the concentration of extracellular nitrate and correlate it with GFP expression. The nitrate portion of the operon consists of the membrane-bound sensor protein NarX and its complementary DNA-binding response regulator NarL. When nitrate is present, NarX will phosphorylate NarL. We engineered this system with pNar, a sfGFP promoter responsive to phosphorylated NarL, so in the presence of nitrate, NarX will phosphorylate NarL, which will initiate the transcription of sfGFP.
Cell-free Implementation
In the 2020 phase of AgroSENSE, we engineered whole-cell nitrate biosensors. However, the Georgia Department of Agriculture raised a concern regarding the use of a cellular biosensor in an agricultural setting. To address any potential safety concerns and stigma around bioengineered products, in 2021, we implemented our biosensor into a cell-free system, eliminating any safety and distribution concerns.
We successfully developed NarX enriched cell-free lysates and tested them with NarX, NarL, and pNar plasmids designed and provided by Dr. Adam Silverman in addition to a variety of nitrate concentrations ranging from 0 ppm to 300 ppm.
Plate-Q is our frugal plate reader capable of quantifying green fluorescence protein (GFP) and Optical Density (OD) from a 96-well standard plate. A typical laboratory plate reader can cost up to $15,000, which is a major bottleneck for smaller scale labs that are unable to afford such equipment. Plate-Q costs under $150 and can be adapted to scan for different types of fluorescent proteins at different wavelengths. Rather than using traditional optical sensors, such as photodiodes found in laboratory microplate readers, Plate-Q takes advantage of a Raspberry Pi camera to capture images of a well plate and extract image features using computer vision and machine learning algorithms. A 440 nm wavelength excitation light is used to measure GFP fluorescence using a 510 nm filter for emission, and a 600 nm light source is used for OD without any emission filter. Plate-Q is completely open-source, so users can customize the design to scan for other fluorescent proteins by replacing the light source and filter for different wavelengths, which retrains the Plate-Q algorithm to take measurements in an affordable, sustainable cost-effective manner.
After obtaining results, we analyzed fluorescence values and determined that Plate-Q works best at greater brightness values (See Figure 8). Because Plate-Q overestimates for lower fluorescence values, this shows that the camera sensor has trouble quantifying lower brightness values (See Figure 8). Moving forward, we plan to utilize either a night-vision camera or the combination of a phone camera and app to better account for Plate-Q’s current inability to quantify lower fluorescence values.
Similar to fluorescence measurement, optical density (OD) in Plate-Q performs more accurately at higher brightness values than lower brightness values. In comparison with a laboratory microplate reader, Plate-Q tends to overestimate optical density (OD) at values approximately greater than 0.3. This is likely due to the low sensitivity of the Raspberry Pi camera sensor at low light settings because higher OD values are associated with less light transmission through a sample or lower brightness values. Due to this, the returned output of Plate-Q will be less reliable at a higher OD. Plate-Q was able to output with an average percent error of 18.74%.
Moving forward, we plan to utilize either a night-vision camera or the combination of a phone camera and app to better account for Plate-Q’s current inability to quantify lower fluorescence values.
LyphoX is our frugal cell freeze-dryer specifically designed to allow easy access to lyophilization for iGEM teams and labs. With the help of Dr. Saad Bhamla from the Georgia Institute of Technology, Lambert iGEM has managed to create a frugal alternative to lyophilizers at approximately one percent of the original cost at about $109 (compared to the original $12,000). An inexpensive lyophilizer would allow these teams and labs to enhance the stability of their cell cultures/extracts, as well as cut down on traditional freezing costs needed during the transportation of these cells. Lambert iGEM has created procedures for both creating and testing using the frugal lyophilizer, making it accessible to anyone looking to assemble their own. We are also currently working on extensive testing and collaboration with Georgia State University, Florida State University, and Johns Hopkins University for proof of concept and functionality.
Overview
Lambert iGEM contacted Sweetwater Urban Farms, a business that grows and sells aeroponics-grown produce in north Georgia, and learned about the prevalence of root rots across hydroponically grown crops. As we tried to determine the cause of these infections, we learned that pre-existing lab kits for pathogen detection were time-consuming and expensive. Furthermore, farmers primarily verify the presence of these diseases in their systems through visual symptoms such as yellowing leaves. However, these signs only appear after prolonged exposure to these pathogens, rendering any form of treatment useless. To make early detection of the pathogens possible, we constructed composite toehold biosensors for two of the most common pathogens, Phytophthora cryptogea and Fusarium oxysporum. These are parts BBa_K3725010 and BBa_K3725020, respectively.
Part Description
Toehold switches are riboregulators that consist of a distinct RNA switch and a complementary trigger. The switch is composed of a hairpin loop structure, the shape of which prevents translation by enclosing the ribosome binding site and start codon. When the trigger sequence binds to the switch, the loop unravels and translation begins, resulting in the expression of a downstream reporter protein. Lambert iGEM chose to utilize toehold switches because of their orthogonality and specificity, which ensures accuracy during detection. The construction of a disease-specific biosensor required us to find a gene unique to the pathogen. When the switch turns on and GFP is expressed, we can confirm that the specific pathogen is present. For the detection of Phytophthora cryptogea and Fusarium oxysporum f. sp. lycopersici, Lambert iGEM focused on the X24 and FRP1 genes, respectively. These genes were selected because they were required for pathogenicity in their respective host organisms and were unique to the species of interest.
The Fusarium Toehold w/ GFP Reporter (BBa_K3725020) is composed of four basic parts: the T7 promoter (BBa_J64997), the switch sequence (BBa_K3725050), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).
The Redesigned Fusarium Toehold w/ GFP Reporter (BBa_K3725022) is composed of four basic parts: the T7 promoter (BBa_J23100), the switch sequence (BBa_K3725080), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).
The T7 Fusarium Trigger (BBa_K3725070) is composed of eight random base pairs, the biobrick prefix, the T7 promoter (BBa_J64997), the trigger sequence (BBa_K3725060), and the T7 terminator (BBa_K731721).
The Phytophthora Toehold w/ GFP Reporter (BBa_K3725010) is composed of four basic parts: the T7 promoter (BBa_J64997), the switch sequence (BBa_K3725050), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).
The T7 Phytophthora Trigger (BBa_K3725040) is composed of four basic parts: the T7 promoter (BBa_J64997), the trigger sequence (BBa_K3725030), and the T7 terminator (BBa_K731721).
Results
To test whether our dual plasmid transformation was successful, we measured and compared the fluorescence and optical density (OD) of the dual plasmid cells, toehold, and pUC19 cells. In order for the dual plasmid transformation to be deemed successful, the fluorescence/OD of the dual plasmid cells should be significantly different compared to that of the toehold, trigger, and pUC19 cells. The obtained measurements of fluorescence/OD for the dual plasmid cells were greatly significantly different (according to SEM bars) from the measurements of the cells transformed with only the toehold and cells transformed with the positive control (pUC19).
Our Advisors
MRS. JANET STANDEVEN
DR. BRITTNEY CANTRELL
Plate-Q
Dr.Saad Bhamla
Dr. Chinna Devarapu
LyphoX
Dr. Saad Bhamla
Dr. Matthew Brewer
Dr. Caesar Rodriguez
Mr. Rajas Poorna
Human Practices
Mr. Clint Crowe
Dr. Bhabesh Dutta
Ms. Megan Heaphy
Ms. Kristen Boscan
Legislation
Commissioner Gary Black
Ms. Natalie Adan
Ms. Ashley Altom
Chairman Todd Jones
PPB
Dr. Rajesh Paul
Ms. Megan McSweeney
Pho
Dr. Mark Styczynski
Ms. Yan Zhang
Ms. Elizabeth Chilton
Dr. Ichiro Matsumura
Nar
Dr. Adam Silverman
Ms. Megan McSweeney
Model
Dr. Mark Styczynski
Ms. Yue Han
Ms. Megan McSweeney
Mr. Clint Crowe
Hardware
In the future, Lambert iGEM hopes to continue improving LyphoX and create a finalized open source model: opening doors for underfunded labs across the globe. Plate-Q hopes to soon go from an open-source camera to using any user’s mobile device for quantification.
Pho
Next year, we plan to continue characterizing our new part BBa_K3725150 in order to improve the fluorescence output and the linearization of our curve. We also plan to expand upon our biosensor efficacy by testing samples from a wider range of concentrations and locations.
Nar
Next year, we plan to continue testing our nitrate sensor with the enriched NarX lysates in order to characterize our biosensor.
PPB
Lambert iGEM is looking at a multitude of proposals to further improve the plant pathogen biosensors. We aim to apply this concept to other common pathogens to assist in the detection of disease in hydroponics systems as well as develop a plant pathogen testing kit containing cell-free extracts. Furthermore, we plan to develop an automatic detection system through machine learning, and research treatment options for infected hydroponics systems. Overall, our goal is to make hydroponics accessible and feasible for all people all over the world, reducing food insecurity.
Modeling
In the future, Lambert iGEM plans to collaborate further with Sweetwater Urban Farms to incorporate the Fusarium epidemiological model into a general purpose hydroponics app to provide farmers with more information. The information could be utilized to extrapolate a Farmer’s number of infected plants at a time where they find that, for example, one of their plants has died. Additionally, more data, such as the plant growth coefficient, will be gathered for the model to further make it more accurate.
Human Practices
In the coming months, Lambert iGEM hopes that the final draft of the regulation dictating the protocols for the distribution and disposal of biosensors will be implemented in Georgia’s agriculture. Through this implementation, we hope that farmers’ skepticism towards the utilization of bacterial biosensors with crops will diminish along with mishandling of bioengineered products.
In order to properly employ the plant-pathogen biosensors, DNA needs to be extracted from Phytophthora cryptogea and Fusarium oxysporum f. sp. Lycopersici, and thus applied to the biosensor. However, similar problems, like ones faced by PPB, arise when DNA extraction is undergone, such as cost, time, safety, and efficiency. As a result, PPB Implementation optimized a protocol to isolate the DNA through sink strainers, to crush and separate the pathogen from the roots, 1X Tae-buffer, to resuspend the cells, RPA, to amplify the DNA in real-world scenarios, capillary tubes, to pipette inexpensively, and OpenCellX, to centrifuge through the entire process. Once the DNA is extracted, it can now bind with the toehold biosensors, and if one of the diseases is present, GFP will be produced, letting users to recognize and take action. Therefore, Implementation creates a frugal and sustainable method, tying together PPB, and allowing practical utilization.
