Team:Lambert GA/Poster

Poster: Lambert_GA

Team

Sachintha Ashok, Abby Bell, Monica Cho, Madhav Gulati, Aryan Gupta, Ruth Hong, Faith Johnson, Tapasya Katta, Saif Khan, Melanie Kim, Krishi Kishore, Anaghaa Kohli, Nikhita Lalwani, Neha Lingam, Sindhu Mettupalli, Zoya Mir, Sri Pinnamareddy, Aditya Prabhakar, Varun Sendilraj, Sahil Sood, Taeyoung Yang, Kathy Ye, Justin Yu

Mrs. Janet Standeven, Dr. Brittney Cantrell

Abby Bell Nar | Pho	Aditya Prabhakar Hardware	Anaghaa Kohli IHP	Aryan Gupta Hardware
Faith Johnson Nutrients	Justin Yu Hardware	Kathy Ye Nar | Video	Krishi Kishore Software
Madhav Gulati Software | Hardware | Wiki	Melanie Kim Nutrients | Collaborations	Monica Cho Nar | Pho	Neha Lingam IHP | Outreach | Wiki
Nikhita Lalwani Nar | Pho	Ruth Hong Nar | Pho | Modeling	Sachintha Ashok Nar | Pho | IHP	Sahil Sood Nar | IHP
Saif Khan Nar | IHP	Sindhu Mettupalli Nar | Pho	Sri Pinnamareddy Outreach | IHP	Taeyoung Yang Nar | IHP
Tapasya Katta Nutrients	Varun Sendilraj Software | Hardware | Wiki	Zoya Mir IHP | Outreach	Dr. Brittney Cantrell Mrs. Janet Standeven

AgroSENSE

Over 23.5 million Americans face food insecurity or have limited access to affordable and fresh produce, leading to nutrient deficiency. Aquaponics is a pragmatic solution to address food insecurity, but maintenance and costs are barriers to implementation. Lambert iGEM’s AgroSENSE utilizes modular hardware, nutrient biosensors, and iOS/Android compatible software to monitor and analyze nutrient levels and environmental conditions to optimize plant growth. Lambert iGEM's Arduino sensors measure environmental conditions while biosensors are utilized to monitor phosphate and nitrate nutrient balance. The team characterized a BioBricks based on E. coli’s native Pho signaling pathway and began cloning to characterize the Nar pathways. The expression of GFP in the presence of these nutrients is analyzed by Fluoro-Q, an improved frugal fluorometer. The data will be quantified by the Agro-Q mobile app, enabling end-users to make informed decisions regarding aquaponics systems. With an emphasis on integration of educational curriculum, AgroSENSE serves as a model for future agricultural biotechnology innovations.

Lambert High School is located in the greater Atlanta area, which is the third-largest food desert in the nation. To address access to nutritious food, Lambert Smart Agriculture, a group of students at Lambert High School experimented with hydroponics and automation to produce efficient, locally sourced vegetables such as kale and lettuce. Hydroponics is a type of agriculture, which is a method of growing plants, usually crops, without soil, by using mineral nutrient solutions in an aqueous solvent. Lambert iGEM's inspiration for this year’s project was sparked by iGEM members who were also in Smart Agriculture. Lambert iGEM members participating in the club pitched the integration of synthetic biology with hydroponics at the team's initial brainstorming meeting in February. Team members discussed common diseases faced in hydroponics systems such as root rot. However, through more in-depth conversations, the team discovered that non-nutrient specific sensors have resulted in Smart Agriculture members estimating the concentrations of the distinct nutrients, phosphate, nitrate, and potassium rather than adding specific fertilizer concentrations based on the existing amount of nutrients in their systems. This led to plant growth variability in their hydroponics systems. In addition, fertilizer solutions add a significant cost to hydroponics. To address this, Lambert iGEM decided to research aquaponics, where fish waste produces many nutrients for plants in conjunction with developing nutrient biosensors, to have a more balanced system.

Figure 1. A few Lambert iGEM members helping Smart Agriculture with their hydroponic greenhouse.
Disclaimer: This image was taken before the COVID-19 pandemic.

Acknowledgements

Our Advisors:
MRS. JANET STANDEVEN
DR. BRITTNEY CANTRELL

*Special thank you to Micheal Choi at Ponix for donating a hydroponics system to our team*

Grant
NSF Grant 1817334

Hydroponics
Mrs. Janet Standeven
Ponix
Mr. Dave Davies and Mrs. Marion Davies

OpenCellX
Dr. Saad Bhamla
Mr. Elio Challa

Fluoro-Q
Dr. Saad Bhamla
Mr. Dan Heieren

Human Practices
CarePointe
Gwinnett Coalition
Georgia Aquaponics
Atlanta Food Bank
APD Urban

Pho and Nar
Dr. Mark Styczynski
Ms. Yue Han
Ms. Yan Zhang
Ms. Elizebeth Chilton

Iron
Dr. Todd Guerdat
Dr. Ichro Matsamura
Dr. Samuel Bell
Mr. Bob Ratajczak

Material Sponsors
MathWorks
IDT
New England BioLabs
SnapGene
Rosco

Hardware

A hydroponics system is required to properly test the efficacy of nutrient production and to test the biosensors and mechanical sensors. The system is composed of a 25-gallon reservoir tank in a vertical hydroponics setup. Other components, such as a swirl filter and fish tank, are unnecessary for a hydroponics system, but they are essential to a successful aquaponics setup. We have designed the next hardware additions and will implement them for next year’s project.

Figure 1. Two of our hardware team members (Justin Yu and Madhav Gulati) assembling the hydroponics system in Lambert High School.

Sensor System:
The Sensor System contains seven sensors that work in conjunction to help monitor plant health and system’s conditions. The first Sensor is the DHT11 Temperature and Humidity sensor, which will be placed outside the system to maximize plant growth using automated responses. The second Sensor will be the CO₂ Sensor. This sensor will track the amount of CO₂ in the atmosphere in 5-10 min intervals as CO₂ vital for plant photosynthesis. The third sensor group, photoresistors, will determine the amount of light the plants will be exposed to then preventing the growth of algae. The fourth is the water temperature probe which is used to measure the water temperature within the system for optimal plant growth. The fifth is the dissolved oxygen sensors which will be used to measure the nutrient solutions content since O₂ levels. Finally, the sixth is the atmospheric O₂ Sensor which will be vital in making sure that the plants are getting the necessary amount of oxygen to stay healthy.
Figure 2. Schematic of the protype electronic system

Figure 3. Our prototype electronics system including a pH sensor, CO₂ sensor, temperature and humidity sensor, and light sensor. All connected by two Arduino microcontrollers.

OpenCellX:
Figure 4. OpenCellX with homogenization module
In addition to our hydroponics hardware and electronics as part of our AgroSense project, Lambert iGEM continued development on the OpenCellX device from the previous year. OpenCellX is a modular, frugal, device capable of replicating the functionality of a tissue-homogenizer, vortex mixer, and centrifuge. The current device utilizes powerful brushless motors and can maintain 2500 Relative Centrifugal Force (RCF) for centrifugation purposes, which in our testing has been sufficient for miniprep similar DNA Extraction protocols. The Tissue-Homogenizer has been tested using Spinach as a model organism, and has shown comparable, yet lower, results to a commercially available device if run for the same amount of time. If run for an additional minute, the results are matched. Both cases, the end product from a DNA Extraction has been more than sufficient for downstream DNA applications. Because of the 3D-printed design and off-the-shelf components, the entire device costs under $65, less than 3% of the cost of a commercial set. Future plans for OpenCellX involve refining the current design to reach higher speeds and test the device under other synthetic biology and molecular biology conditions.

Figure 5. OpenCellX in homogenization mode operating at 750RPM compared to a commercial tissue-homogenizer operating at 300hz to lyse a sample of spinach tissue. Results were quantified through DNA extraxtion and measured using a NanoDrop.
Figure 6. OpenCellX in the centrifugation setting operating at 2800RCF compared to a commercial centrifuge operating at 12,000RCF in a bacterial MiniPrep. 4 minutes were added to each step on OpenCellX to account for the slower speed.

Modeling

Before characterization in the lab, we built a deterministic Ordinary Differential Equation model of the Pho Regulon signaling pathway with part BBa_K2447000. Using the model, we were able to better understand the signaling pathway and also simulated GFP expression of the phosphate biosensor to explore the sensitivity of the BioBrick.


Figure 1. Diagram of the ODE model in MATLAB Simbiology software.
The model’s predictions followed the same trend as NUS Singapore 2017 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 NUS Singapore measured that of an entire culture of cells.


Figure 2. Model simulation of GFP expression for 0-100uM phosphate concentrations.

Pho

Background
Phosphorus is one of the most important nutrients for plant survival. In aquaponics systems, the main source of phosphorus is inorganic phosphate; monitoring its concentration is often expensive, time-consuming, and imprecise. In order to combat these issues, we cloned and characterized part BBa_K2447000, an extracellular phosphate sensor with GFP reporter, in the plasmid pSB1C3 for potential use as a biosensor within aquaponics systems.

Part Description
GFP expression of the BioBrick is activated by the PhoB transcription factor from the phosphate-sensitive Pho Regulon signaling pathway that naturally exists in E. coli. When the biosensor detects lower concentrations of extracellular phosphate, it expresses higher levels of GFP; conversely, when it detects higher concentrations of phosphate, it expresses lower levels of GFP.


Figure 1. Pho Regulon signaling pathway activating GFP expression of part BBa_K2447000

Characterization Curve
Our team constructed a characterization curve that will later be used as a reference for aquaponics users to determine their system’s phosphate level. We measured GFP expression of the biosensor under phosphate concentrations from 0uM to 100uM, a range of phosphate typically found within aquaponics systems.
Our characterization data followed the trend of GFP expression predicted by our model except for the 100uM phosphate concentration. In later troubleshooting, we found that this error was due to improper dilution of phosphate media; our team could not conduct another set of testing due to time constraints.

Figure 2. Characterization curve for GFP Expression vs. Phosphate Concentration
Proof Of Concept
In the coming year, we plan to demonstrate how our phosphate biosensor cells would work to identify unknown phosphate concentrations in water samples from aquaponics systems. We will measure the GFP expression of our biosensor cells with these samples, and determine the corresponding phosphate concentration based on our characterization curve. Using a commercial test kit to measure actual phosphate concentrations in these samples, we will validate the accuracy of our detection method.

Nar

Background:

The Nar biosensors are split into two components: the NarL system, which measures nitrate, and the NarP system, which measures nitrite and nitrate. This year, Lambert iGEM is primarily focusing on the NarL system, which is based on the HKUST-Rice 2015 project; next year, the novel NarP part will work with Lambert's NarL part to calculate the corrected difference and concentrations for both nutrients.

Nar Systems:
Figure 1: Model detailing the NarL signaling pathway.
Figure 2: Model depicting the NarP signaling pathway.
Cloning:

Lambert iGEM is working on cloning the NarL part through Gibson Assembly and has successfully PCR both the insert, which was split into two fragments, and the pSB1C3 backbone.

Figure 3. Lambert iGEM’s Gibson Assembled NarL construct.
Figure 4. Lambert iGEM’s Gibson Assembled NarP construct.
Figure 5. Gel depicting amplified Nar Fragments (Fragment 1 and Fragment 2) and pSB1C3 Backbone (Backbone 1 and Backbone 3).
Engineering Success:
Figure 6. Diagram detailing the engineering success workflow for Nar, starting from the research to experimental design to cloning to future plans.
Future Plans:
In the coming months, Lambert iGEM is continuing to clone NarL and NarP and plans on conducting trials to characterize their sensitivity to differing concentrations of nitrate and/or nitrite. We are also researching cell-free nitrate and nitrite sensors- a potential direction for our project’s second year in order to ensure the safety of our system.

Quantification

The quantification of GFP molecules from the biosensors is important for the determination of nutrient levels. Fluoro-Q is an improved fluorometer built upon Lambert iGEM 2019’s FluoroCents device. The basic premise of a fluorometer is that excitation energy is used to excite GFP molecules within a biological sample. The energy that is emitted from these molecules is then passed through an emission filter and quantified with a detector.

Figure 1. Overview of Fluoro-Q device and schematics (A) Fundamental schematic of how fluorescence quantification works in a fluorometer including key components like an excitation filter, emission filter, light source, and detector (B) Animated model of Fluoro-Q device with smartphone camera, glass emission filter, and excitation light (C) Top view of Fluoro-Q device with power source, wiring, and 3D-printed components (D) Side view of GFP sample from perspective of emission filter (E) Side view of Fluoro-Q device from perspective of excitation light source
Figure 2. Quantification images of two-fold serial dilutions of Fluoro-Q taken on iPhone XS ready for ImageJ image processing pipeline
Fluoro-Q uses an excitation wavelength LED of 450 nanometers specific to the fluorescent reporter of choice to excite the molecules. The energy that is emitted in the form of light is then passed through a glass emission filter that improves the specificity of light filtration. These components are enclosed within a 3D-printed casing. The camera of a smartphone is used to capture a raw image of the sample. This image is then passed through an image quantification pipeline in ImageJ that determines the intensity of fluorescence from the sample relative to a negative and positive control.

Figure 3. Data from two-fold serial dilution comparing optical density to level of fluorescence measured using Fluoro-Q

Lunchbox Microscope:
In addition to Fluoro-Q, we worked on creating a fluorescent attachment to the Lunchbox Microscope, a portable brightfield digital microscope developed by Lake Region Optics to promote STEM engagement that is capable of connecting to mobile devices and computers to capture microscopic images. To adapt this technology to suit the needs of our project, we developed an attachment to create a fluorescent microscope using LEDs, filters, and 3D-printed components. The final design of the Lunchbox Microscope attachment thus utilized a blue light LED chip with wavelength range 450-460nm alongside a glass emission filter of wavelength 510nm. The light source was positioned perpendicularly to the digital camera in the Lunchbox Microscope. Testing with a fluorescent liquid protein sample resulted in successful visualization of fluorescence under the Lunchbox Microscope with the fluorescent attachment developed by Lambert iGEM. This was compared to visualizations under a traditional laboratory fluorescent microscope.

Figure 4. Side-by-side comparison of fluorescent liquid protein sample visualization under fluorescent microscope and Lunchbox Microscope

Apps

AgroSENSE:
The AgroSENSE app is a complete guide for users to build and maintain a hydroponics system. Users are given a step-by-step video and written instructions to build their ideal system. In addition, the app also contains maintenance guides for hydroponics and healthy cooking recipes.
The AgroSENSE mobile app is built to help users set up their own hydroponics system with ease. Users first take a survey to generate their ideal systems by taking into account their specific parameters: plants, size, budget, and various other factors.Upon completion of the survey, users will be given written instructions and a tutorial video to start the building process.

Figure 1. Flow chart demonstrating layout of the AgroSENSE app and how a user navigates the app.


Agro-Q:
The Agro-Q mobile app has a wide variety of features that allow users to maintain their hydroponics system. Users can view sensor data of pH, water temperature, CO₂, atmospheric temperature, and light intensity, via the app, and analyze plant growth with models on the app. The Agro-Q app allows collaboration among the user by sharing their specific hydroponics setups.
The Agro-Q app is divided into 4 sections: map, sensor data, models, and monitor. On the map, users can locate local hydroponics, allowing for a sense of community through the app. The sensor data tab shows historical and real time data from the various electronic sensors throughout their system. The data will be refreshed periodically and updated in the app. Users can also generate statistical models to analyze the growth of plants based on the different growth conditions and factors of the system. Overall, this app allows remote users to keep track of the different components in the system.

Figure 2. Flow chart demonstrating layout of the AgroQ app and how a user navigates the app.

Human Practices

Integrated Human Practices
Lambert iGEM collaborated with a variety of stakeholders including food banks, commercial and local aquaponics producers, and local schools to determine if AgroSENSE is good and responsible for society. Inspiration: Our project was inspired by Smart Agriculture, a group of students at our high-school who experimented with hydroponics and automation to provide nutritious, efficient, locally sourced produce such as kale and lettuce. A few Lambert iGEM members also participated in this club and proposed integrating aquaponics and synthetic biology. This inspired our initial investigation of biosensors to address nutrient balance concerns within alternative agriculture systems.

Figure 1. A few Lambert iGEM members helping Smart Agriculture with their hydroponic greenhouse build.

Problem:
Dr. Guerdat, an environmental engineer at the USDA and author of Recirculating Aquaculture, informed us of the laborious procedures in maintaining aquaponics systems, specifically maintaining nutrient balance. For his large scale aquaponics system, he told us that he had to send his samples to a laboratory and the ion measurement instruments were costly. This shifted our focus to developing nitrate, nitrite, and phosphate biosensors as well as researching possible bioremediation of Iron from fish waste sludge, informing users of the nutrient levels in their system.

Figure 2. Phosphate and Nitrate deficiency in plants leads to debilitated plant growth.

Development:
Dr. Mark Styczinski from the Georgia Institute of Technology and Dr. Ichiro Matsumura from Emory University guided us in the development of our biosensors. They helped troubleshoot concerns in Nar and Pho regulons’ inability to measure certain thresholds of nutrients and the interference of the native regulons with our synthesized parts. Meanwhile, Dr. Saad Bhamla from the Georgia Institute Technology mentored us in developing our low cost bead homogenizer OpenCellX, an improvement on our last year’s hardware.

Proposed Implementation:
We consulted with Gwinnett Coalition and Atlanta Community Food Banks to discuss the potential to address food insecurity. They believed aquaponics to be an effective solution to provide nutritious food, however, noted that the public's lack of familiarity may be a barrier to implementation. We created the AgroEATs cookbook based on common aquaponically grown produce to promote awareness. Initially, we intended to implement the AgroSENSE system in Atlanta’s food desert communities. However, conversations with APD Urban, an urban planning organization, sparked concerns about biosensors distributed in the public, so we shifted our focus from community based systems to school groups and biotechnology teachers. This way trained teachers will be primarily responsible for safely maintaining and discarding biosensor cells. We were able to connect with high school biotechnology teachers across Georgia and developed plans to implement AgroSENSE in schools through Georgia Bio, a non-profit aspiring to advance scientific knowledge within schools.

Figure 3. Lambert iGEM members discussing how they can use AgroSENSE to supplement Georgia’s high-school agriculture standards

Figure 4. Lambert iGEM pitching our AgroSENSE Educational Integration idea to biotechnology teachers like Ms. Cynthia Greer and receiving feedback.

Science Communication

Workshops:
Lambert iGEM partnered with multiple high schools in Ghana by creating practical science workshops to help prepare students for international science exams. Members gave lectures, held online workshops, and led practical science experiments that students could perform within their own homes. These workshops focused on biology and biotechnology principles, with an emphasis on the importance of frugal science. In order to accomodate a larger audience, we gathered volunteers around Georgia to translate the material into Hindi, Korean, and Chinese, which we plan on distributing to underfunded classrooms in which these are the primary languages as part of our two year project. After our workshops, we conducted a series of surveys and assessments to evaluate the students’ growth in both knowledge and interest in iGEM and biotechnology. The results displayed a significant increase in understanding. In an effort to make science more accessible, all of our resources are open-source.
Figure 5. Lambert iGEM members teaching workshop attendees how to build a foldscope.

AgroEats:
Lambert iGEM created a cookbook: “AgroEats,” available free-of-charge on the AgroSENSE app. AgroEats demonstrates how aquaponic systems can be incorporated into various lifestyles by displaying culturally diverse recipes. Working families can choose from over 25 simple, nutritious recipes utilizing aquaponically grown produce and common household ingredients. The recipes are all based on Feeding America Nutritional Guidelines as provided by the Atlanta Community Food Bank.

Infographics:
In response to the coronavirus pandemic, many schools are now shifting to a digital format. To accommodate this change for students, Lambert iGEM created a series ranging from basic biological concepts to complex synthetic biology procedures. Upon the completion of the infographics, Lambert iGEM began distribution to high school biology and biotechnology teachers in Forsyth County, Georgia in order to elicit feedback regarding classroom use. All surveyed teachers were impressed with the concise nature of the infographics, but some believed they were better suited for honors students, and requested more introductory-level topics. In response, Lambert iGEM drafted a list of requested topics to cover in infographics as part of the two-year project.

Achievements

Bronze:

• Competition Deliverables: Wiki, videos, poster
• Attributions: Members, collaborators, sponsors
• Project Description: AgroSENSE aims to address food insecurity by integrating aquaponics and synthetic biology
• Contribution: Pho Characterization Curve on BBa_K2447000, an extracellular phosphate sensor with GFP reporter

Silver:

• Engineering Success: Development of Nar nitrate/nitrite biosensor
• Collaboration: 2020 University of Rochester, Queens University, Hainan China, GreatBay_SCIE, FDR-HB Peru, CCA_San_Diego, Korea_HS, and ICS_BKK
• Human Practices: Education and engagement
• Proposed Implementation: Working with teachers, food banks, and urban planning firm to develop plans on integrating AgroSENSE systems across Georgia.

Gold:

• Integrated Human Practices: integrated feedback from experts in Synthetic Biology, Hardware, and Aquaponics, and Nonprofits to develop AgroSENSE and contributed to promoting awareness of synthetic biology and aquaponics within our community.
• Project Modeling: Deterministic Ordinary Differential Equation model of the Pho Regulon signaling pathway with BBa_K2447000
• Proof of Concept: theoretical phosphate sample testing
• Science Communication: held practical science workshops with 3 high schools in Ghana in which members of Lambert iGEM covered various topics in biology over 4 weeks; also developed a cookbook and supplemental biotechnology infographics for use by community members and teachers respectively.
• Excellence in Another Area: OpencellX, frugal substitute for other lab equipment

Future Directions

Hardware:
In the future, Lambert iGEM will work to integrate the two environments it has been developing separately, the hydroponics and aquatic, into one self sustaining aquaponics ecosystem. Further, the current electronics layer will be improved upon to incorporate additional sensors and also use devices like water heaters, pH buffers, aerators, and fans to autonomously maintain certain key conditions through Arduino control.

Software:
Next year, Lambert iGEM will attempt to develop an app to streamline the quantification process for Fluoro-Q without the need for manual image processing. The process will be integrated into the hydroponics system setup so that samples can automatically be extracted and quantified for periodic nutrient measurements. The AgroSense and Agro-Q apps will be further developed to include more collaborative features and ability to analyze more complex data points for intelligent monitoring.

Pho:
Next year, Lambert iGEM plans to test our proof-of-concept by utilizing the phosphate biosensor and characterization curve to identify phosphate concentrations of water samples from aquaponics systems.

Nar:
In the coming months, Lambert iGEM is continuing to clone NarL and NarP and plans on conducting trials to characterize their sensitivity to differing concentrations of nitrate and/or nitrite. We are also researching cell-free nitrate and nitrite sensors- a potential direction for our project’s second year in order to ensure the safety of our system.

Modeling:
In the coming year, the team aims to create deterministic Ordinary Differential Equation models for both NarL and NarP systems of the Nar Operon. With the model, Lambert iGEM hopes to better understand the systems and simulate GFP expression of the NarL and NarP BioBricks.

Human Practices:
In the coming year, Lambert iGEM hopes to foster stronger communication with communities within food deserts, broadening our perspective on the individual challenges faced by the residents, as well as consulting governmental representatives. Additionally, we aim to collaborate with teachers around the world to incorporate aquaponics and biotechnology into their local curriculums.