Team:Lambert GA/Implementation

PROPOSED IMPLEMENTATION

PROJECT OVERVIEW

Our project AgroSENSE aims to address the issues of food insecurity in urban areas. The lack of fresh produce available along with the expensive consumer prices of these items lead to concerns over agricultural sustainability. While hydroponics, a compact and modular form of agriculture, produces nutrient-dense and high-yield crops, its implementation in urban communities is often impeded due to the difficult maintenance of small-scale systems that often leads to nutrient fluctuations thus declining plant health. To combat these barriers and increase the frequency of hydroponics use, AgroSENSE provides an accurate and efficient method of nutrient monitoring and pathogen detection that can also be safely distributed to our end users. We propose to utilize a frugal lyophilizer to freeze-dry cell-free lysates and bacterial samples of our phosphate and nitrate biosensors and Phytophthora cryptogea and Fusarium oxysporum f. sp. lycopersici toehold switches, which can later be used in conjunction with our frugal plate reader for fluorescence quantification (see Fig. 1).

Figure 1. Overview of Proposed Implementation and how it allows for our project to be applied into the real world.


PROPOSED END USERS

AgroSENSE encourages anyone who is in need of locally-sourced, nutritious food to utilize hydroponics systems by increasing the systems’ accessibility and sustainability. Specifically, the nutrient-dense crops grown through hydroponics are beneficial for heavily-populated, indoor urban communities affected by food insecurity since they cannot implement traditional agriculture. Our relatively low-cost nutrient biosensors provide users with specific nutrient concentrations, eliminating errors arising from commercial test strips and maintaining the systems’ stability. The pathogen toehold switches can detect plant pathogens very early, while they are still treatable, thus allowing farmers to retain a larger crop yield.

BIOSENSORS

Implementation into the Real World

Phosphate Sensor (Whole Cells):

  1. Receive lyophilized biosensor cells, instructions, and other disposal materials from distributor (e.g. UV light, bleach, and ethanol).
  2. Collect a 10 mL sample of water from the hydroponics system.
  3. Rehydrate lyophilized bacterial samples with 30μL of sterile water, then spread them on an LB agar plate with 25mg/mL chloramphenicol antibiotic for 24 hours.
  4. Inoculate a colony of cells into 5mL of sterile LB and 25mg/mL chloramphenicol antibiotic and let them grow overnight, shaking at 170RPM at 37 degrees Celsius, then resuspend in nutrient-deficient media (e.g. MOPS).
  5. Add 500μL of hydroponics water sample to the 5ml culture and incubate, shaking at 170RPM at 37 degrees Celsius, for 2-3 hours for GFP expression.
  6. Utilize frugal plate reader, PlateQ, to measure OD600 and fluorescence (485 nm/528nm) values.
  7. Compare fluorescence/OD600 value of the samples to the provided characterization curve and determine concentration.
  8. Multiply concentration by 10 in order to account for the diluted sample during Step 5.
  9. Dispose all cells appropriately, as instructed by our biosensor use regulation (See Lambert_GA 2021: Integrated Human Practices).

Nitrate Sensor and Plant Pathogen Toehold Switches (Cell-Free Lysates):

  1. Receive lyophilized lysate, instructions, and other disposal materials (e.g. bleach, and ethanol).
  2. Collect a 10 mL sample of water from the hydroponics system.
  3. Rehydrate lyophilized lysate with 34 μL of hydroponics water sample.
  4. Incubate the lysate, shaking at 170 RPM at 37 degrees Celsius, for 3 hours for GFP expression.
  5. Utilize frugal plate reader, PlateQ, to measure fluorescence (485 nm/528 nm) values.
  6. Compare the fluorescence value of the samples to the control and determine the nutrient concentration or the presence of pathogen.
  7. Dispose of all materials appropriately, as instructed by our proper biosensor use regulation (See Lambert_GA 2021: Integrated Human Practices).

*For data demonstrating our project’s likeliness to work in a real setting, see Lambert_GA 2021: Proof Of Concept*

Safety

Successfully designed and characterized biosensors are meaningless without the ability to be safely and cheaply transported to our end users. Traditionally, cells are difficult to transport due to their delicate nature and often pose a biosafety threat to the larger community. The lyophilizer is responsible for freeze-drying our biosensors for longer shelf life and safer distribution. Through the utilization of sub-zero temperatures and a near-perfect vacuum to sublimate water out of cell samples, freeze-drying preserves cells in an inactive state until rehydration. Compared to many immobile commercial lyophilizers that often cost tens of thousands of dollars, our frugal $109 substitute, LyphoX (see Fig. 2), allows our proposed end users to have access to our biosensors and thus nutrient-rich and high-yield hydroponics crops.

Figure 2. Picture of our frugal lyophilizer, LyphoX.


Furthermore, we reached out to the Food and Safety Division of the Georgia Department of Agriculture and to Georgia Bio, a non-profit organization dedicated to increasing the development of Georgia’s life sciences. After discussing the safety of our biosensors during various virtual conference calls, we realized that there are no current regulations in place to mitigate an outbreak caused by biosensors or bioengineered products. This year, our team has been collaborating with these stakeholders to script a regulation that outlines a safe, effective way of distributing and disposing of agricultural biosensors.

PLANT PATHOGEN DNA EXTRACTION

While our toehold switches showed success in detecting the presence of plant-based pathogens, we also found that the current DNA extraction methods are expensive, lab-work-intensive, and time-consuming as evidenced by the usage of ELISA or PCR and the need to ship diseased plant samples to labs [1]. In order to provide hydroponics users with the ability to perform their own DNA extraction with cost-effective equipment, we developed a method for extracting pathogenic material from root samples, the main entry-point for Phytophthora cryptogea and Fusarium oxysporum f. sp. Lycopersici.

Implementation into the Real World

  1. Receive or supply the necessary materials for extracting Phytophthora and Fusarium DNA from plants’ roots ((See Lambert_GA 2021: PPB)
  2. Cut part of the roots (around 2 inches length) and place inside between two sink strainers.
  3. Place enclosed strainers over the beaker and grind until significant root volume has been crushed and no more liquid is excreted into the beaker.
  4. Pour 40 mL of 1X TAE through the mesh sink strainers, making sure to pour over the crushed root sample. Pour resulting liquid in a Falcon Tube.
  5. Let the particles settle to the bottom and remove the supernatant.
  6. Add 400μl of distilled water to particles and shake vigorously.
  7. Pour all of the resulting supernatant into a 1.5 mL microcentrifuge tube and centrifuge using OpenCellX for 10 minutes in 2 minute intervals at 5000 RPM.
  8. Incubate the DNA solution at 65 degrees celsius with a DIY sous vide for an hour or overnight at 4 degrees Celsius (in the fridge).
  9. Use a capillary tube to draw up 1 μl of DNA solution (first line) and release (place on and press) into the lid of RPA Mastermix.
  10. Use another capillary tube to draw up 2.5 μl of MgOAc and release into the lid of RPA Mastermix (separate from other solution).
  11. Let the DNA incubate at 37-42 degrees Celsius for 30-40 minutes.
  12. Finally, add the DNA to the toehold biosensor(s). A green fluorescence will indicate the presence of either Fusarium or Phytophthora, depending on which toehold was applied.

Protocol for Separating Fusariumand/or Phytophthora from Roots Materials to Be Supplied by the User Per Test 1.5ml microcentrifuge tubes: ($0.35 for 5) 1 Falcon Tube ($0.83 for 1)Capillary Tubes ($0.01 for 2)40 mL 50mM EDTA (pH 8.0) ($0.05)Sous Vide (DYI using online tutorials)2 Mesh Sink Strainers ($1.60 for 2 → reusable)Premade RPA mastermix and MgOAc ($2.22 for 1)Toehold Biosensor ($0.50 for 1)OpenCellX ($65.00 for 1 unit → reusable) PROTOCOL 1. Cut part of the roots (around 2 inches length) and place inside between two sink strainers2. Place enclosed strainers over the beaker and grind until significant root volume has been crushed and no more liquid is excreted into the beaker.3. Pour 40 mL of 1x TAE through the mesh sink strainers, makingsure to pour over the crushed root sample. Pour resulting liquid in Falcon Tube 4. Let the particles settle to the bottom and remove liquid.5. Add 400μl of distilled water to particles and shake vigorously.6. Pour all of the resulting mixture into a 1.5 mL microcentrifuge tube Centrifuge using OpenCellX for 10 minutes in 2 minute intervals at 5000 RPM.7. Incubate the solution at 65 degrees celsius with a DIY sous vide for an hour or overnight at 4°C (in the fridge).8. Use capillary tube to suck 1 μl of solution (first line) and release (Place on bulb and release) into the lid of RPA Mastermix. 9. Use another capillary tube to suck 2.5 μl of MgOAc and release into lid of RPA Mastermix (separate from other solution)10. Let the solution incubate at 37-42 degrees celsius for 30-40 minutes.11. Finally, apply this solution to the toehold biosensor(s).12. A green fluorescence will indicate the presence of either Fusarium or Phytophthora depending on which toehold was applied.

Figure 3. A protocol that hydroponics users can easily follow to extract and detect pathogen presence.


Safety

While the Tris Acetate EDTA (TAE) we supply is only 1X concentration, it can cause respiratory and skin irritation if exposed. Thus, we recommend wearing gloves, long sleeves and pants, as well as masks to protect users from possible harm. Additionally, wearing this personal protective equipment, such as gloves, can reduce contamination of the DNA leading to purer DNA and better results.

*For data demonstrating our project’s likeliness to work in a real setting, see Lambert_GA 2021: Proof of Concept*

REFERENCES

[1] Boriyo, H. (2019, July 19). Plant Pathology Diagnostic Laboratory Services. OSU Extension Service. Retrieved October 20, 2021, from https://extension.oregonstate.edu/harec/plant-pathology-diagnostic-laboratory-services.