Team:Lambert GA/Pho

PHO

BACKGROUND

For a plant to grow and function properly within a hydroponics system, phosphate levels need to be maintained within specific concentration ranges of 50µM to 100µM. From our 2020 team’s research, discussion with experts, and experience with hydroponics, we learned that inorganic phosphate levels are difficult to precisely detect and maintain using commercial test kits as they are often costly, time-consuming, and inaccurate [1]. To address these issues, we developed our two-year project, AgroSENSE, centered on designing and characterizing a low-cost, highly-sensitive phosphate biosensor. Our characterization curve provides an alternative for hydroponics users to accurately and conveniently detect specific phosphate concentrations within a system. To address biosafety concerns, we propose to lyophilize, or freeze-dry, our biosensor cells for public distribution.

Figure 1. Diagram of the Pho Regulon signaling pathway.


To design the biosensor, we utilized the natural E. coli Pho Regulon signaling pathway (see Figure 1), which responds to extracellular phosphate levels [2]. Inorganic phosphate (Pi) initiates the pathway by passing through a PhoE porin protein. High levels of Pi inhibit the histidine kinase PhoR by repressing its autophosphorylation, which prevents PhoR from phosphorylating the PhoB transcription factor and results in little to no downstream transcription of the Pho Regulon genes [3]. Inversely, low levels of Pi allows for active phosphorylation of the PhoB transcription factor and subsequent transcription of the native genes [4]. For more information about the Pho Regulon and its response to different phosphate concentrations, please refer to the Pho Background section of the 2020 Lambert iGEM page.

PART DESIGN

BBa_K2447000

BBa_K2447000, created by NUS Singapore iGEM 2017 as an improvement to BBa_K116404, is an extracellular phosphate sensor with a PhoB promoter and a GFP reporter.

Figure 2. Diagram of the BBa_K2447000 construct.

As shown in Figure 2, BBa_K2447000 consists of an inducible promoter (BBa_K116404) 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.

Additional Parts

In the process of improving upon our initial characterization curve, we created 3 new parts: BBa_K3725110, BBa_K3725120, and BBa_K3725150. However, replacing the PhoB promoter with the PhoA (alkaline phosphatase gene) promoter and its consensus sequences ultimately did not refine the characterization curve. For more information on the new parts and their subsequent characterization curves, please refer to their respective Parts Registry pages or the Engineering Success page.

CHARACTERIZATION

Initial Characterization

Purpose of Characterization

Through literature review, our 2020 team discovered that phosphate concentrations between 50-100µM are ideal for plant growth in hydroponics systems. For detailed characterization, we tested GFP expression of our biosensor on the extracellular phosphate level range of 0-150µM.

Protocol

  1. Grow biosensor cells in Erlenmeyer flasks with 60mL LB and 60µL antibiotic for 24 hours shaking at 170 RPM.
  2. Ensure that the cells are at an OD600 value between 0.4 and 0.8 using a spectrophotometer.
    • If OD600 is less than 0.4, grow cells for longer, checking OD600 every 20-30 minutes.
    • If cells have grown too much, dilute to desired OD600 with LB.
  3. Add 5mL of the cell culture into a 15mL liquid culture tube.
  4. Centrifuge for 10 minutes, discard the LB supernatant, and resuspend the cell pellet in 5mL MOPS media.
  5. Add 500µL of diluted phosphate solution.
  6. Grow biosensor cells for another 2-4 hours.
  7. Pipette 150µL of biosensor cells into a well plate to measure cell density (OD600) and fluorescence values using a plate reader.


Fluorescence/OD600 Characterization Phosphate Concentrations (µM) 0 0 50000 100000 150000 200000 30 60 90 120 150 F/OD600

Figure 3. Graph of our initial BBa_K2447000 characterization, including the abnormal bump.



As shown in Figure 3, the characterization curve revealed an inverse relationship between phosphate concentrations and fluorescence/OD600, consistent with expectations from the Pho Regulon. However, an abnormal bump appeared around 120µM of extracellular phosphate and remained constant throughout multiple rounds of testing and troubleshooting.

Modifications

Characterization Range

After reaching out to Dr. Ichiro Matsumura from Emory University and Dr. Mark Styczynski from Georgia Institute of Technology, we decided to focus on a more accurate and detailed characterization for phosphate concentrations under 100µM.

Different and Mutated Promoters

We also extensively researched different biological parts and sequences that could potentially improve our curve. We replaced the PhoB original promoter with the following two promoters:

  • PhoA Promoter (BBa_K1682012) - The inducible promoter, created by HKUST-Rice 2015 iGEM, was originally made to control expression of the PhoA alkaline phosphatase gene after activation by the phosphorylated PhoB transcription factor.
  • PhoB Consensus Promoter (BBa_K3725130) - We engineered the promoter by replacing parts of the original sequence with the PhoB recognition consensus sequence, a calculated order of the most repeated nucleotides found at each position in an alignment of multiple other sequences that have a similar function [5].

However, characterization using the different and mutated promoters exhibited little to no fluorescence compared to our initial data, leading us to focus on the other modifications instead. For more details on the new promoters and their subsequent characterization curves, please refer to their respective Parts Registry pages or the Engineering Success page.

Characterization Protocol

The failed characterization trials with the modified promoters led us to revisit our characterization protocol. While the purpose of the MOPS media resuspension step was to eliminate the effect of extracellular phosphate on our biosensor cells, we realized that the trace of residue LB supernatant may still affect the data. Additionally, we noticed that resuspension may not be enough to ensure that the culture being transferred to a well plate had an even distribution of biosensor cells. To resolve these issues, we decided to add MOPS wash steps prior to adding the phosphate solutions and a vortex step prior to transferring the culture to a well plate. The modified protocol is as follows:

  1. Grow biosensor cells in Erlenmeyer flasks with 70mL LB and 70µL antibiotic for 24 hours shaking at 170 RPM.
  2. Ensure that the cells are at an OD600 value between 0.4 and 0.8 using a spectrophotometer.
    • If OD600 is less than 0.4, grow cells for longer, checking OD600 every 20-30 minutes.
    • If cells have grown too much, dilute to desired OD600 with LB.
  3. Add 5mL of the cell culture into a 15mL liquid culture tube.
  4. Centrifuge for 10 minutes, discard the LB supernatant, and resuspend the cell pellet in 1mL MOPS media.
  5. Transfer 300µL of the resuspension into a 1.5mL microcentrifuge tube. Centrifuge for 2 minutes, and discard the MOPS media supernatant.
  6. Add 300µL of MOPS media and centrifuge again for 2 minutes. Discard the MOPS media supernatant.
  7. Resuspend the cell pellet in 1mL MOPS media.
  8. Add 100µL of diluted phosphate solution.
  9. Grow biosensor cells for another 2-4 hours.
  10. Pulse vortex the biosensor cells.
  11. Pipette 150µL of biosensor cells into a well plate to measure cell density (OD600) and fluorescence values using a plate reader.

Characterization Curve

Using the modified phosphate range and characterization protocol, we collected data for the BBa_K2447000 construct. The characterization curve of the biosensor (Figure 4) exhibited a significantly greater amount of fluorescence and a stronger linear negative trend throughout all phosphate concentrations ranging from 0-100μM. This data closely parallels the predictive ODE model created last year (Figure 5) and the previous characterization data by NUS Singapore iGEM 2017.



Phosphate Concentrations (µM) 0 0 100000 200000 300000 400000 500000 20 40 60 80 100 F/OD600 F/OD vs Phosphate Concentrations

Figure 4. Characterization curve for BBa_K2447000 for phosphate concentrations between 0-100µM.




0 0 2x10-12 4x10-12 6x10-12 8x10-12 200 400 600 800 1000 Phosphate Concentrations (uM) GFP Expression (Molecules) Deterministic ODE ModelSimulation of GFP Expression

Figure 5. Prediction of relationship between GFP expression and phosphate concentrations between 0-100µM made by deterministic ODE model.


TESTING

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:

  1. Dick Creek: between 0-10µM
  2. Chattahoochee Pointe Park Lake: between 0-10µM
  3. 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 6) and identified each samples’ phosphate concentrations. The results were as follows:

  1. Dick Creek: 1µM
  2. Chattahoochee Pointe Park Lake: 0µM
  3. 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 6. Phosphate concentration analysis for Dick Creek (left) and Lambert Hydroponics (right) using the Lamotte testing kit.


Lyophilization

Our phosphate sensor heavily utilizes membrane-bound proteins found within the Pho Regulon signaling pathway. This dependence prevents the sensor from being developed into a cell-free system, making lyophilization a potential solution for safe distribution to the public.

Using LyphoX, we freeze-dried our phosphate biosensor in order to determine whether our cells still function properly after lyophilization. As shown in Figure 7, the biosensor was expressing GFP both before and after lyophilization, demonstrating that LyphoX did not compromise the cell structure of the samples. Additionally, successful sequencing results, as shown in Figure 8, display that the freeze-drying process did not contaminate or denature the DNA (See: Proof of Concept).


Figure 7. Visible GFP expression of our phosphate sensor before (left) and after (right) lyophilization.


Figure 8. Sequencing results of our phosphate sensor aligned with the original sequence.

REFERENCES

[1] Laboratory Evaluation of Ion-Selective Electrodes for Simultaneous Analysis of Macronutrients in Hydroponic Solution. Publication: USDA ARS. (n.d.). Retrieved October 20, 2021, from https://www.ars.usda.gov/research
/publications/publication/?seqNo115=253882.

[2] Santos-Beneit, F. (2015). The Pho regulon: a huge regulatory network in bacteria. Frontiers in Microbiology, 6. https://doi:10.3389/fmicb.2015.00402.

[3] Uluşeker, C., Torres-Bacete, J., García, J. L., Hanczyc, M. M., Nogales, J., & Kahramanoğulları, O. (2019). Quantifying dynamic mechanisms of auto-regulation in Escherichia coli with synthetic promoter in response to varying external phosphate levels. Scientific Reports, 9(1). https://doi:10.1038/s41598-018-38223-w.

[4] Crépin, S., Chekabab, S., Bihan, G. L., Bertrand, N., Dozois, C. M., & Harel, J. (2011). The Pho regulon and the pathogenesis of Escherichia coli. Veterinary Microbiology, 153(1-2), 82-88. https://doi:10.1016/j.vetmic.2011.05.043.

[5] Huang, T.-W., Wen, S.-Y., Chang, C.-Y., Tsai, S.-F., Wu, W.-F., & Chang, C.-H. (2012, October 5). Genome-Wide PhoB Binding and Gene Expression Profiles Reveal the Hierarchical Gene Regulatory Network of Phosphate Starvation in Escherichia coli. PLOS ONE. https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0047314.