Team:Lambert GA/PPB

PPB

OVERVIEW

During the maintenance of our hydroponics system in the 2020 competition season, Lambert iGEM struggled to control root rot infections in our plants. To assess the prevalence of these diseases in other hydroponics systems, we spoke to Mr. Crowe of Sweetwater Farms and learned that he struggled to contain widespread infections of root rot plant pathogens, validating the need for a solution. This problem is widespread: a survey taken by the American Aquaponics Association and the EU Aquaponics Hub indicated that 84.4% of users observed plant disease symptoms in their aquaponics systems and 78.1% were unable to determine the causal agent [1]. Farmers primarily verify the presence of these diseases through visual signs such as yellow leaves. However, symptoms often arise after an extended period of exposure, so visual detection of the pathogen indicates the plants have already sustained significant damage. Furthermore, there are limited options and scientific literature available for plant pathogen management and current detection methods such as serological testing kits are expensive and time-consuming [2]. These factors can lead to inappropriate treatment and crop loss. To combat this obstacle in pathogen detection, Lambert iGEM designed Phytophthora and Fusarium pathogen toehold switch biosensors as an effective and rapid alternative to traditional methods. We decided to focus on the Phytophthora and Fusarium genera, two of the most common genera responsible for root rot pathogens found in hydroponics and similar systems [3].

HOW TOEHOLD SWITCHES WORK

Toehold switches are riboregulators that consist of a distinct RNA switch and a complementary trigger. The RNA switch is composed of a hairpin loop structure that 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, thus ensuring accuracy during detection.

Figure 1. General Mechanism of Toehold Regulation of Downstream Reporter Gene.

PART DESIGN

Lambert iGEM constructed disease-specific biosensors by using a gene unique to each respective pathogen for the trigger sequence. The presence of the trigger sequence would allow a ribosome to attach and express GFP. Because the trigger sequence is derived from a gene specific to the pathogen of interest, GFP expression allows us to 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. We selected these genes because they were required for pathogenicity in their respective host organisms and were unique to the species of interest. We obtained these sequences via UniProt, an online database of protein sequences.

Once we obtained the sequences of the genes of interest, we designed toehold switch-trigger pairs using NUPACK (Nucleic Acid Package) software with code created by Takashi et. al. that was shared by Ms. Megan McSweeney of the Styczynski Lab of the Georgia Institute of Technology.

Figure 2. Trigger-toehold switch structure derived from NUPACK for Phytophthora cryptogea.

Figure 3. Trigger-toehold switch derived from NUPACK for Fusarium oxysporum.

Figure 4. BBa_K3725020: Fusarium Toehold w/ GFP Reporter Construct.

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).

Figure 5. BBa_K3725070: T7 Fusarium Trigger Construct.

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).

Figure 6. BBa_K3725010: Phytophthora Toehold w/ GFP Reporter.

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).

Figure 7. BBa_K3725040: T7 Phytophthora Trigger Construct.

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).

Figure 8. BBa_K3725022: Improved Fusarium Toehold w/ GFP Reporter.

The Redesigned Fusarium Toehold w/ GFP Reporter (BBa_K3725022) is composed of four basic parts: the T7 promoter (BBa_J64997), the switch sequence (BBa_K3725080), a GFP reporter (BBa_E0040), and the T7 terminator (BBa_K731721).

EXPERIMENTAL DESIGN

To test the functionality of our switch and trigger sequences, we ran a dual-plasmid transformation with both plasmids of a toehold and trigger pair. The cells should fluoresce green when both plasmids are present. We utilized electrocompetent transformation because of its high transformation efficiency. We transformed pUC19 DNA along with our dual plasmid to act as a positive control for growth, in addition to transforming cells with just the switch plasmid and just the trigger plasmid to confirm that the cells would not fluoresce without the presence of both sequences.

Electrocompetent Transformation Protocol

Required Materials:

4 new cuvettes (chilled), 4 initial tubes, 4 final tubes, SOC (prewarmed), 2.5uL micropipette, 20uL micropipette, 1000uL micropipette, + (tips), waste beaker, 4 plates (prewarmed), centrifuge, incubator, electroporation machine

  1. Thaw electrocompetent cells and place SOC and plates into 37℃ incubator
  2. Pipette 50 uL cells each into 1 mm gap of 5 chilled cuvettes
    3. Positive Control:
    - 1uL pUC19 DNA    		 TR:
    - 1uL Trigger DNA    		 TH:
    - 1uL Toehold DNA    		 Dual Plasmid:
    - 1 uL Toehold DNA - 1 uL Trigger DNA    		 Dual Plasmid 2:
    - 1 uL Toehold DNA - 3 uL Trigger DNA
  3. Add plasmids
  4. Pipet up and down gently INTO gap, make sure none overlapping
  5. Electroporate one cuvette at a time, record voltage and time
  6. Immediately add 950 uL of 37℃-warmed SOC from incubator
  7. Pipet SOC up and down and put mixture into a new (warm) microcentrifuge tube
  8. Place into a shaking incubator for an 60 minutes at 250 rpm
  9. Plate 150 uL of liquid onto plate

In order to test whether our dual plasmid transformations were successful, we had to measure and compare the fluorescence and optical density (OD) of both of our dual-plasmid transformations, both toehold transformations, the positive control transformation, and plain growth media. In order for our dual plasmid transformations to be deemed successful, the dual-plasmid cells must have a statistically significant difference in fluorescence/OD compared to that of the other measured groups.

The dual plasmid cells were grown in culture tubes containing 5mL of LB and 5ul of both kanamycin and carbenicillin; the toehold cells and pUC19 cells were grown in culture tubes containing 5mL of carbenicillin LB; the trigger cells were grown in 5 mL of kanamycin LB. After overnight incubation, the cells were resuspended in water. Before testing, we first vortexed the liquid culture tubes to ensure the cells are evenly distributed throughout the cultures to minimize variation between each measurement. The liquid cultures were pipetted into well plates, with 150uL increments of the liquid cultures placed in each well. The fluorescence and OD were quantified using a plate reader, and then the measured fluorescence and optical density of water was subtracted from all data values to account for background fluorescence. The fluorescence divided by optical density was calculated for all data points, and bar charts with SEM error bars were created.

RESULTS

After successfully transforming the redesigned Fusarium pair and the original Phytophthora pair, we performed a miniprep, restriction digest, and ran a gel to confirm that the cells uptake both the toehold and trigger plasmids. We induced liquid cultures with IPTG to initiate transcription when the cells reached an OD of 0.5 and promptly proceeded to resuspend in water. After multiple trials to obtain sufficient amounts of data, we created an SEM graph bar based off of plate reader data (fluorescence/OD) and observed a significant difference between the dual plasmid and the control variables (toehold only, pUC19).

Sample Type Dual Plasmid Toehold pUC19 0 20 40 60 80 Fluorescence/OD Comparison of Improved Fusarium Fluorescence/OD Values by Sample Type(With IPTG)

Figure 9. Mean fluorescence/OD of IPTG-induced Fusarium pair 2 dual plasmid transformation compared to toehold and pUC19 with SEM error bars. Ran at gain of 40.



Sample Type Dual Plasmid Toehold pUC19 0 25 50 75 100 150 125 175 200 225 Fluorescence/OD Comparison of Phytophthora Fluorescence/OD Values by Sample Type(With IPTG)

Figure 10. Mean fluorescence/OD of IPTG-induced Phytophthora dual plasmid transformation compared to toehold and pUC19 with SEM error bars. DP stands for dual plasmid, TH stands for toehold only. Ran at gain of 60.

IMPLEMENTATION

Process

To utilize the toehold biosensors, samples must be taken from the hydroponics system, lysed, and amplified to ensure ample DNA yield for the analysis of pathogenic presence. As mentioned before, Phytophthora cryptogea and Fusarium oxysporum are responsible for causing root rot in hydroponics systems and are spread through the roots. For this reason, Lambert iGEM decided to extract pathogen genomic DNA from root samples. Compared to alternative methods, root samples have higher pathogen concentrations, which correlates with higher pathogen DNA concentrations, further solidifying the use of root samples for toehold input [1]. Because of the impracticality of the current methods, we ultimately pursued more sustainable, efficient, and frugal methods of extraction through the roots of the plants (see Fig. 11).

Overview

Crush roots between stacked sink strainers and pour TAE Add water and centrifuge with OpenCellX to isolate pathogen DNA Pour out TAE and isolate pathogen Transfer DNA to new tube RPA DNA Amplification

Figure 11. The diagram shown above is an overview of our process as to how pathogen DNA is extracted from infected root samples. Our process follows a 3 pronged process for DNA extraction: Lysis (where the cells are ground and crushed using sink strainers), Purification (where TAE is poured and centrifuged using OpenCellX to isolate unnecessary organic matter), and Amplification (where DNA is amplified using RPA, a frugal process that ensures a proper DNA yield for the toehold biosensor to utilize).

Components

Lysis

To properly lyse the cells, we used sink strainers to sustainably crush roots and pathogen cells, exposing the genetic material of the pathogen. The sink strainers are designed as per an interlocking mechanism using the grates of the top sink strainer to emulsify the root sample held in the bottom one, thus, breaking down the cell wall and membrane using applied torque and friction between the two surfaces.

Figure 12. Utilizing sink strainers to hold and crush roots, breaking down the pathogen cell membranes and nuclear membranes.

Purification

After the samples are lysed, the samples undergo a purification step to further isolate the pathogenic DNA from the root samples through the usage of Tris-Acetate-EDTA(TAE). TAE is crucial for resuspending and is used after the cells are crushed using the sink strainers. TAE is able to permeate the cell wall, leading to better yields and stable DNA that can be used further along in the extraction [2]. Afterward, TAE is poured out and DNA is suspended in water, as well as centrifuged to separate from unnecessary organic materials and cell residue, until amplification.

Amplification

DNA extracted from an infected root sample is generally present in low quantities, posing a problem for toehold detection. In order to ensure an ample yield of DNA for the toehold biosensor to detect, the extracted DNA needs to have the toehold trigger sequence amplified. Methods using PCR amplification require the use of expensive equipment (i.e. thermocycler) , which can cost upwards of $500 [3]. Thus, we employed an isothermal process of Recombinase Polymerase Amplification (RPA).

$ LOW COST CONSTANT TEMPERATURE LOW TIME SELECTION SPECIFICITY $2.22 37° C - 42° C 30 - 40 Minutes Detections in Impure Samples A G C G A T T A G C C G T A T C

Figure 13. Advantages of RPA for amplification [4].


By combining MgOAc (the solution that starts RPA) and the extracted DNA with the premade mastermix (contains enzymes and primers that correspond with our trigger), farmers can feasibly amplify the plant pathogen DNA, which contains the trigger sequence for each toehold.

Biosensor

After our DNA sequence is amplified, cell-free lysates, which house the toehold biosensor, convert the Fusarium and/or Phytophthora target DNA to RNA if pathogen DNA is present. The RNA then binds with the toehold resulting in the downstream expression of GFP.

Frugal Engineering

Sink Strainers

To frugally lyse our cells, we used conventional sink strainers that contain minuscule grates, which will help shear the cell wall, and expose the genetic material. To maximize the area of contact between the roots samples and grates, we utilized an interlocking pattern of mesh strainers that uses torque force as well as applied pressure to simultaneously crush during hand rotation.

Centrifugation/Vortexing: OpenCellX

We decided to utilize OpenCellX, a frugal homogenizer/centrifuge that was previously developed by Lambert iGEM during the 2019 & 2020 seasons. OpenCellX helps vortex and centrifuge components at a relatively high speed (RPM of about 5000) to adequately pellet solid residue of cells and proteins and thoroughly mix components and reagents. This device was also used to start the RPA reaction.

Capillary Tubes

To eliminate the usage of a conventional pipette, we looked into a frugal alternative that can take minute volumes of solutions: capillary tubes and bulbs. Capillary action is utilized to uptake small volumes of liquids from 1 to 200 microliters. The bulb is attached after intaking the volume and, when pressed, releases the volume of liquid. This aims to eliminate the necessity for expensive and difficult-to-use pipettes in the RPA process, which requires pipetting of liquids at 1 and 2.5 microliters, making extraction easier for farmers.

Microliters vs Millimeters Traveled on Capillary Tube Millimeters 3.82x - 0.469 R2 = 0.98 uL 0 0 5 10 15 20 1 3 5 4 2 mm

Figure 14. Capillary tube experimentation data. This measures the volume of liquid (uL) in comparison to distance traveled on capillary tube (mm).

Experimentation was carried out to confirm whether these could provide a consistent and reliable drawing of solutions. Hence, 12 trials were conducted comparing the consistency of distance traveled as a fill line would denote where to draw a solution to. There is a strong positive correlation between distance traveled and volume retrieved by a pipet, meaning that the capillary tubes show a promising future for needed reliability and frugality. Parametric equations could be derived to further improve the accuracy of a fill line to a desired volume by taking into account density, draw speed, tube angling, and experimentation.

Figure 15. Video shows usage of frugal pipette to take 2.5 microliters of dye. Taken by Ricky Jiang.

Experimentation

Overview

In order to demonstrate that DNA could be extracted using these methods and to further optimize our process, we conducted a series of experiments utilizing yeast, a safer but viable alternative for initial testing since yeast and Fusarium are both fungi and Phytophthora is a fungus-like protist [5].

Incubating the Samples

To begin our experimentation, we added Baker’s yeast to the hydroponically grown lettuce samples and let them incubate for 12-18 hours. This allows the roots to absorb yeast cultures, thus “infecting” the plant. (see Fig. 16).

Figure 16. Pictures of hydroponic lettuce samples “infected” with yeast.

Optimizing our process

After our samples were incubated, we lysed, purified, and amplified our samples using our theoretical process. Initially, we based our purification process on the Wizard Genomic DNA Purification Kit Protocol and utilized more expensive equipment such as thermocyclers, microcentrifuges, vortexers, etc [6]. To help formulate our extraction protocol, and cut down costs, we systematically eliminated each of these reagents and eventually began utilizing frugal equipment and Recombinase Polymerase Amplification using the Liquid Basic Protocol.

Final Protocol

The below is the protocol that was followed to extract genomic DNA from fungi and fungi-like protists:

  1. Receive or supply the necessary materials for extracting Phytophthora and Fusarium DNA from plants’ roots.
  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.

Results

After we formulated our protocol through systemic testing, we began experimenting on Fusarium, which required significant Biohazard Protocols regarding growing our samples and disposing of them (See Lambert_GA 2021: Safety). Using commercially available samples from Agdia, we infected our root samples, and a culture tube (for our positive control), with Fusarium oxysporum. We then let the Fusarium samples grow for 48 hours.

Figure 17. Plants infected with Fusarium before extraction (left) and after (right). The left plant shows minimal symptoms, and does not include many of the major signs of infection such as root rot, as the one on the left.

After we extracted our samples (as per the theoretical process mentioned above), we ran an agarose gel electrophoresis and checked for band presence, intensity, and length.

Figure 18. Agarose gel of our extracted and amplified pathogen DNA. Lanes 7 and 8 (circled) represent our sample tested with our optimized process from root samples, Lane 4 serves a control where we tested our optimized process on a pure culture tube of Fusarium. Lanes 2-3 display our optimized solution with just a minor substitution of PCR instead of RPA. Lane 1 serves as a control of yeast.

As demonstrated by our gel electrophoresis, we have indicated the presence of our bands in wells 1, 4, 6, and 7. This provides solid proof for our extraction process and displays the extracted DNA, which our recently developed Fusarium and Phytophthora toehold biosensors can now utilize. The absence of bands of 2 and 3 justifies our theoretical method as it displays the non-amplification of our samples through PCR, and provides further evidence for the specific selectivity of RPA, which can amplify through complex samples of Proteins and DNA, as shown by the presence of faint bands of 450-600 kb on wells 6 and 8 (The presence of bands on the bottom of our gel displays excess nucleotides, which are commonly seen in unpurified samples of RPA Liquid Basic).

Practical Applications

Figure 19. A protocol hydroponics users can follow to extract and detect pathogen presence.

Overall, we intend to provide pre-prepared aliquots to the farmer for usage and optimal testing, all arranged in a single array of tubes. The aliquoted solutions are as follows.

  1. TAE (40 mL in Falcon Tube)
  2. RPA Mastermix (46.5 uL in PCR Tube)
  3. MgOAc (2.5 uL in PCR Tube)

These solutions will be moved amongst various tubes via the capillary tube pipette that we constructed.

Benefits

Our process will have several benefits when compared to conventional methods including:

  • Frugality: Our process costs $4.05 per test (one-time cost of OpenCellX ($65) and Sink Strainers ($1.60)). Conventional methods meanwhile cost upwards of $70 per test[7].
  • Speed: Our process takes ½ - 1 day to complete and extraction and detection can be done at the hydroponics system. Meanwhile, conventional methods can take upwards of 5-7 days[7].
  • Sustainability: Combined with the toehold switch biosensor, our process can detect infection samples before symptoms of root rot occur, which meets United Nations 11th Sustainability Goal: Sustainable Cities by providing high yields of safe, nutrient-dense, and healthy foods to all people who live in cities[8].

FUTURE GOALS

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, which will contribute to our goal of increasing crop yields and building sustainable cities. Eventually, we plan to develop a plant pathogen testing kit containing cell-free extracts of biosensors for various pathogens for accessibility and practicality. To add to our plant disease detection process, we hope to develop a monitoring system for the automatic detection and treatment of plant disease using machine learning. Finally, we will look into potential treatment options for plant pathogens, granting users the ability to resolve issues without relying on costly and time-consuming professional remedies. Overall, our goal is to make hydroponics accessible and feasible for all people all over the world, reducing food insecurity.

REFERENCES

PPB Toehold References

[1] Stouvenakers, G., Dapprich, P., Massart, S., & Jijakli, M. H. (2019). Plant pathogens and control strategies in aquaponics. Aquaponics Food Production Systems, 353-378.

[2] Almeida, R. P. P. (2019, June 22). Emerging plant disease epidemics: Biological research is key but not enough. PLOS Biology. Retrieved October 20, 2021, from https://journals.plos.org/plosbiology/article?id=10.1371%2Fjournal.pbio.2007020.

[3] Rivas-García, T., González-Estrada, R. R., Chiquito-Contreras, R. G., Reyes-Pérez, J. J., González-Salas, U., Hernández-Montiel, L. G., & Murillo-Amador, B. (2020). Biocontrol of phytopathogens under aquaponics systems. Water, 12(7), 2061.

[4] Green, Alexander A., et al. “Toehold Switches: De-Novo-Designed Regulators of Gene Expression.” Cell, vol. 159, no. 4, 6 Nov. 2014, pp. 925–939., https://doi.org/10.1016/j.cell.2014.10.002.

PPB Implementation References

[1] Djalali Farahani-Kofoet, R., Witzel, K., Graefe, J., Grosch, R., & Zrenner, R. (2020, June 24). Species-specific impact of fusarium infection on the root and shoot characteristics of asparagus. Pathogens (Basel, Switzerland). Retrieved October 20, 2021, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350344/.

[2] Chauhan, D. T., & Chauhan, A. T. A. D. T. (2020, January 10). Importance of tris-EDTA (TE) buffer in DNA extraction. Genetic Education. Retrieved October 20, 2021, from https://geneticeducation.co.in/importance-of-tris-edta-te-buffer-in-dna-extraction/.

[3] The $499 open source PCR machine / thermal cycler. OpenPCR. (2019). Retrieved October 20, 2021, from https://openpcr.org/#:~:text=OpenPCR%20%2D%20the%20%24499%2%20Machine%20%2F%20Thermal%20Cycler.

[4] Recombinase Polymerase Amplification, or RPA, is the breakthrough, isothermal replacement to PCR. TwistDx. (2020). Retrieved October 20, 2021, from https://www.twistdx.co.uk/en/rpa.

[5] Olsen, J., & Nelson, P. (2011, April 25). Phytophthora infestans. microbewiki. Retrieved October 20, 2021, from https://microbewiki.kenyon.edu/index.php
/Phytophthora_infestans.

[6] Wizard® Genomic DNA Purification Kit Technical Manual. Wizard® Genomic DNA Purification Kit Protocol. (n.d.). Retrieved September 22, 2021, from https://www.promega.com/resources/
protocols/technical-manuals/0/wizard-genomic-dna-purification-kit-protocol/.

[7] 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.

[8] United Nations. (n.d.). Goal 11 | Make cities and human settlements inclusive, safe, resilient and sustainable. United Nations. Retrieved October 20, 2021, from https://sdgs.un.org/goals/goal11.