PROOF OF CONCEPT

BIOSENSORS

Phosphate Sensor

Phosphate concentrations between 50μM and 100μM are ideal for plant growth in hydroponics systems. Our team characterized BBa_K2447000, a phosphate sensor that utilizes the Pho Regulon signaling pathway, through detailed characterization of GFP expression in response to the extracellular phosphate level range of 0μM to 100μM. As shown in Figure 1, The characterization curve of our biosensor exhibited a strong linear negative trend throughout all phosphate concentrations, which closely paralleled the predictive Ordinary Differential Equations model (Figure 2) created by our 2020 team.

Figure 1. Characterization curve for BBa_K2447000 for phosphate concentrations between 0-100µM.

Figure 2. Prediction of relationship between GFP expression and phosphate concentrations between 0-100µM made by deterministic ODE model.

Nitrate Sensor

Similarly, the ideal nitrate concentration for hydroponics plants is between 40 ppm and 160 ppm. Our nitrate sensor utilizes the native Nar Operon in E.coli; we characterized BBa_K3725210 (pNar promoter) shared from the Jewett research group at Northwestern University by measuring its GFP expression in response to phosphorylated NarL, which is produced by NarX in the presence of nitrate. This year, we prepared NarX enriched cell-free lysates (Figure 3), and began initial experimentation. Using NarX, NarL, and pNar plasmids designed by Dr. Adam Silverman, we experimented with nitrate concentrations between 0 ppm and 300 ppm.

Figure 3. NarX enriched cell-free lysates that will be experimented with NarX, NarL, and pNar plasmids.

Phytophthora and Fusarium Toehold Switches

Lastly, we proved the efficacy of our Fusarium and Phytophthora plant pathogen toehold switches by conducting a dual plasmid transformation with their complementary triggers. As shown in Figures 4-7, our dual plasmid transformations were successful and its GFP expression was higher than that from the transformed toehold alone. This demonstrates that in the presence of specific plant pathogens, our toehold switches can successfully detect and subsequently show fluorescence.

Figure 4. Gel confirming successful dual plasmid transformation of Phytophthora toehold switch and trigger. Wells 3 and 6 are standardized DNA ladders, and wells 1, 2, 4, and 5 contain the Phytophthora toehold and trigger at 2,800bp and 2,000bp, respectively.

Figure 5. Gel confirming successful dual plasmid transformation of Fusarium toehold switch and trigger. Well 5 is the standardized DNA ladder, and well 6 contains the Fusarium toehold and trigger at approximately 2,800 bp and 2,000bp, respectively.

Figure 6. Graph showing significantly higher GFP expression in dual plasmid transformation compared to transformation of only the toehold.

Figure 7. 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.

In order to ensure that plant pathogens can be isolated, lysed, and amplified easily by our end users, we developed a cost-effective, frugal process for DNA extraction (See: PPB). We utilized Saccharomyces cerevisiae, baker's yeast, as a safer alternative to pathogenic Phytophthora cryptogea and Fusarium oxysporum for initial testing.

Our proof of concept involved infecting lettuce roots with yeast to test our extraction process on harmless fungal DNA. As shown in the gel results from Figure 8, yeast DNA was successfully extracted from our artificially infected lettuce root samples using our optimized process. To further validate our procedure, we successfully infected lettuce roots with Fusarium oxysporum, and extracted the DNA from the infected root samples (Figure 9), thus solidifying the success of our plant pathogen biosensors if they were to be implemented into the real world.

Figure 8. Gel confirming successful extraction of yeast DNA with our optimized process. Well 5 contains a standard DNA ladder, and wells 3, 4, 6, 7, and 8 contain the yeast DNA amplified through RPA.

Figure 9. Gel confirming successful extraction of Fusarium DNA with our optimized process. Well 7 contains the Fusarium DNA amplified using RPA.

CELL-FREE LYSATES

Cell-free systems allow for cellular processes to occur in vitro, eliminating both the physical constraints of a living cell and the safety hazard of live cell distribution. This is achieved through the preparation of a lysate that removes genomic DNA and the cell membrane, resulting in a solution with isolated cell parts later used for synthesis. In order to address the biosafety issues regarding the public distribution of cells, we proposed to develop cell-free lysates of our nitrate sensor and plant pathogen toehold switches.

We successfully prepared a lysate from BL21(DE3) competent E.coli cells (Figure 10) and another enriched by NarX (Figure 11) as a proof of concept to demonstrate the efficacy of our lysate preparation protocol.

Figure 10. BL21(DE3) competent E.coli cell-free lysates

Figure 11. NarX enriched cell-free lysates

We initially experimented with a plain lysate and a plasmid with a T7 promoter driving sfGFP transcription provided by Dr. Adam Silverman. The data revealed that sfGFP was produced in the presence of our BL21(DE3) competent E.coli cell lysate and master mix, as shown in Figure 12 below.

Figure 12. Plate Reader fluorescence data for T7 GFP and T7 GFP + 10mM IPTG

After our initial experimentation yielded successful results, we tested the same lysate with our Fusarium and Phytophthora toehold and trigger plasmids. Preliminary tests did not reveal a statistical difference between the baseline and in the presence of plasmids. We are continuing to troubleshoot our experimentation protocols to successfully implement both plant pathogens into a cell-free system.

In addition, we began testing our NarX enriched lysate with nitrate concentration between 0 and 300 ppm in conjunction with the NarX, NarL, and pNar plasmids, again provided by Dr. Silverman. We expect our enriched lysate to express varying concentrations of sfGFP depending upon the concentration of nitrate. As the concentration of nitrate increases, the concentration of sfGFP should also increase.

LYOPHILIZATION

Since our phosphate sensor is not able to be developed into a cell-free system due to its heavy utilization of membrane-bound proteins within the Pho Regulon signaling pathway, lyophilization is key for its safe distribution to our end users. 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 $109 LyphoX device is frugal and can easily be replicated by potential users.

As shown in Figure 13, the negative control (See: Hardware) grew no colonies while the experimental group exhibited adequate growth of lyophilized cells after rehydration, proving that LyphoX did not compromise their cellular structure.

Figure 13. Negative control group (left) in comparison to experimental group (right) after being left out in the same room for 1 day.

We also lyophilized E. coli cells and saw success in sublimating an average of 97.58% of the water weight over ten trials, illustrating our device’s effectiveness in numerical data (Figure 14). Meanwhile, Figure 15 below shows the physical results of our lyophilized cells after successful runs of the standardized procedure, indicating that the qualitative end results were consistent with our expected observations: solid, flaky cell matter left behind.

Trial Number	Initial Weight (g)	Final Weight (g)	% Water Sublimated
1	0.767	0.650	97.5%
2	0.766	0.646	100%
3	0.770	0.663	89.2%
4	0.766	0.656	91.7%
5	0.766	0.650	96.7%
6	0.766	0.651	95.8%
7	0.770	0.650	100%
8	0.767	0.649	98.3%
9	0.773	0.653	100%
10	0.768	0.650	98.3%

Figure 14. Table of data collected from LyphoX tests. *Percent water sublimated was calculated by dividing the weight change by 0.120 (30μL x 4 = 120μL = .120 grams)

Figure 15. Pictures of LyphoX test results after successful completion of the standardized procedure (from left to right: trials 3, 9, and 10)

As shown in Figure 16, the commercial lyophilizer (Labconco Modulyo-D) was successful in freeze-drying an average of 98.68% of the water weight. Although its percent water sublimated was greater than that of LyphoX, this shows the effectiveness of our frugal device and its standardized procedure in replicating the quantitative data of a commercial lyophilizer.

Trial Number	Initial Weight (g)	Final Weight (g)	% Water Sublimated
1	0.744	0.624	100%
2	0.737	0.618	99.2%
3	0.748	0.629	99.2%
4	0.740	0.623	97.5%
5	0.746	0.629	97.5%

Figure 16. Table of data collected from commercial lyophilizer Labconco Modulyo-D tests. *Percent water sublimated was calculated by dividing the weight change by 0.120 (30μL x 4 = 120μL = .120 grams)

Furthermore, we freeze-dried our phosphate biosensor in order to prove that our sensors still function properly after lyophilization. As shown in Figure 17, we confirmed that our biosensor was expressing GFP both before and after lyophilization. Additionally, our sequencing results (Figure 18) proved that the biosensor DNA was not damaged or altered during the freeze-drying process.

Figure 17. Visible GFP expression of our phosphate sensor before (left) and after (right) lyophilization.

Figure 18. Sequencing results of our phosphate sensor aligned with the original sequence.

SAMPLE TESTING

To implement our biosensors into the real world, we experimented with our cells using local water sources, including samples from Dick Creek and Chattahoochee Pointe Park in Forsyth County, Georgia, as well as hydroponics samples from the Sweetwater Aeroponics System.

Our phosphate sensor and its characterization curve indicated that the water samples had the following phosphate concentrations:

Dick Creek: between 0-10μM
Chattahoochee Pointe Park Lake: between 0-10μM
Sweetwater Aeroponics System: between 60-70μM

To compare our data to that of a commercial kit, we also utilized the Lamotte phosphate testing kit (Figure 18) and identified each samples’ phosphate concentrations. The results were as follows:

Dick Creek: 1μM
Chattahoochee Pointe Park Lake: 0μM
Sweetwater Aeroponics System: approximately 50μM

The similar results between analysis with our phosphate sensor and a commercial test kit further substantiated that our biosensor can accurately detect extracellular inorganic phosphate levels, validating its usefulness in regulating hydroponics systems.

Figure 19. Phosphate concentration analysis using our water samples and the Lamotte testing kit.

FLUORESCENCE QUANTIFICATION

Lambert iGEM has also designed a frugal plate reader, PlateQ, to quantify fluorescence readings from our biosensors, aiding in the identification of exact nutrient concentrations or the detection of specific plant pathogens. PlateQ will provide its consumers a frugal, cost-effective, and simple fluorescence-reading device.

The goal of our device is to allow for fluorescence quantification through a smartphone application rather than a Raspberry Pi camera. Though PlateQ has not yet reached this stage, data from testing proves that this goal is plausible. As shown in Figures 20 and 21, the average percent error of PlateQ is less than 4.7% for fluorescence values of 600-2000, and the average standard deviation is 2.187. This shows that PlateQ is able to accurately quantify fluorescence and optical density in comparison to a traditional and commercial plate reader.

Figure 20. Image of well plate with different concentrations of fluorescent dye.

Figure 21. Comparison of fluorescence in plate reader and Plate-Q at different concentrations and ranges.

Team:Lambert GA/Proof Of Concept