Team:Lambert GA/Pho
BACKGROUND
The most essential nutrients to plant function are phosphorus, nitrogen, and potassium, as well as trace concentrations of other elements [3]. Fertilizer is used to concentrate such nutrients and allow plants to slowly absorb them through their roots. In aquaponic systems, nitrogen is produced via nitrogenous fish waste, and phosphorus is sourced from the addition of phosphate rocks. However, the method to determine phosphorus and nitrogen concentrations in the circulating water is inefficient. Aquaponic farmers often rely on qualitative symptoms such as wilting, purple coloring, and dark spots to identify specific nutrient deficiencies. This causes a feedback gap as plants take several weeks to respond to a lack of nutrients; by the time farmers identify the absent nutrient(s), it may be too late to correct. Chemical test strips and electrical testing are also common, but require regular maintenance and can be expensive, costing between $500 and $3,000 to purchase. Furthermore, testing kits that require external analysis add costs and time to ship samples and receive data. To combat these obstacles facing aquaponic farmers today, Lambert iGEM characterized an extracellular phosphate biosensor paired with a hardware/software system to provide a more accurate, timely, and cost effective method of detecting and monitoring nutrients.
As mentioned above, plant cells must have access to a specific concentration of inorganic phosphate (Pi), an important source of the element phosphorus. Some uses of phosphorus in plants include the elongation and division of cells, starch/sugar transfer, and root health and growth [4]. In order to uptake Pi, cells go through a series of reactions - including signaling pathways - in the extracellular space, membranes, and cytoplasm.
Lambert iGEM aims to use E. coli, with natural signaling pathways, to create a biosensor which detects extracellular substances such as Pi. Similar to the general phosphate signaling pathway in plant cells, E. coli bacteria have a phosphate signaling pathway to regulate the intake and concentration of inorganic phosphate.
PHO REGULON SIGNALING PATHWAY
E. coli bacteria have a naturally occurring phosphate-sensitive signaling pathway to control expression of the Pho Regulon, which responds to extracellular inorganic phosphate levels and transcribes regulatory genes [1]. The signaling pathway, shown below, is initiated once Pi (inorganic phosphate) molecules enter the cell by passing through PhoE porin proteins in the outer membrane. In the periplasmic space, Pi binds to the protein PstS, which carries Pi to the PstABC transporter complex located on the inner membrane. The PstABC complex consists of the PstA/C transmembrane channel and the permease PstB, which phosphorylates PstA/C to actively transport Pi across the inner membrane. Different levels of Pi within the cytoplasm will then bind to the accessory protein PhoU and consequently activate or deactivate transcription of Pho Regulon genes.
Figure 1. Diagram of the Pho Regulon signaling pathway.
Research has shown that higher levels of Pi in the cytoplasm deactivate the transcription of Pho Regulon genes [2]. When Pi is available in the cytoplasm, it binds to the accessory PhoU protein. The bound PhoU-Pi complex inhibits the PstB permease, preventing PstA/C from further transporting Pi into the cytoplasm. The same PhoU-Pi complex also inhibits the histidine kinase PhoR by repressing its autophosphorylation. Through this process, PhoR is unable to phosphorylate, or activate, the transcription factor PhoB. PhoB is inactive, and therefore unable to activate transcription of the Pho Regulon, so the genes of the Pho Regulon are not expressed. Over time, Pi dissociates from PhoU - therefore restarting the cycle.
On the other hand, lower levels of Pi limit the accessory PhoU protein from binding to Pi; PhoU is therefore unable to inhibit the permease PstB. This allows Pi to enter the cytoplasm through the transmembrane channel PstA/C. Because of the initial lower levels of Pi, there is no PhoU-Pi complex to inhibit the histidine kinase PhoR. PhoR autophosphorylation occurs, and then PhoR phosphorylates the PhoB transcription factor. Once activated, PhoB binds to the promoter region of the Pho Regulon and transcription of genes within the regulon is initiated; these genes translate into the various proteins involved in the signaling pathway.
Essentially, lower levels of extracellular phosphate result in expression of the Pho Regulon genes, and higher levels lead to less expression of those genes.
PART DESIGN
BBa_K2447000
Part BBa_K2447000, an extracellular phosphate sensor with GFP reporter, was created by NUS Singapore iGEM 2017 as an improvement to the part BBa_K116404, an existing external phosphate sensing reporter. While NYMU Taipei 2008 initially created part BBa_K116404 with a medium-strength RBS (BBa_B0032), NUS Singapore 2017 increased the sensitivity of the phosphate sensor by replacing BBa_B0032 with a stronger RBS, BBa_B0034.
Figure 2. Part design construct of BBa_K2447000.
As shown in Figure 2, BBa_K2447000 consists of a PhoB-activated promoter (BBa_K116401), strong RBS (BBa_B0034), GFP reporter (BBa_E0040), and double terminator (BBa_B0015).
The promoter BBa_K116401 is activated by the PhoB transcription factor from the Pho Regulon signaling pathway that exists naturally in E. coli. In part BBa_2447000, natural Pho Regulon genes of the Pho signaling pathway are replaced by the GFP reporter BBa_E0040 so that activation of the promoter via binding of phosphorylated PhoB can result in expression of GFP.
In conditions of high extracellular phosphate, the histidine kinase PhoR is inhibited and is unable to phosphorylate PhoB, making it unable to activate the transcription of the downstream GFP reporter. On the contrary, in conditions of low extracellular phosphate, PhoR is active and is able to phosphorylate PhoB, resulting in transcription of the downstream GFP reporter.
Figure 3. Diagram of part BBa_2447000 under different levels of extracellular phosphate.
According to literature, the Pho Regulon signaling pathway has a threshold of 4uM of extracellular phosphate [6]. In other words, extracellular phosphate concentrations greater than 4uM should result in no transcription of GFP while concentrations less than 4uM should result in transcription of GFP.
CHARACTERIZATION
PURPOSE OF CHARACTERIZATION
Based on Lambert iGEM’s ODE model, the 4uM threshold of BBa_K2447000 seems to be less strict than described above. Extracellular phosphate concentrations greater than 4uM, even in the range of 100uM, will still result in minimal GFP expression. To improve upon the existing characterization of the phosphate sensor, Lambert iGEM tested with a greater number of phosphate concentrations from 0 to 100uM in intervals of 20uM.
See Drylab: Model to read more about Lambert iGEM's deterministic ODE model.
EXPERIMENTAL DESIGN
In order to improve part BBa_K116404 created by NYMU-Taipei 2008, NUS Singapore iGEM 2017 created part BBa_K2447000 by replacing the weaker RBS BBa_B0032 with a stronger RBS BBa_B0034. This allowed the BBa_K2447000 extracellular phosphate sensor to be much more sensitive to various phosphate concentrations from 0uM to 1000uM.
Through research, the team discovered that typical phosphate levels in aquaponics systems range from 10ppm to 40ppm [3]. Using dimensional analysis, the team concluded that maintaining a phosphate concentration between 50uM and 100uM is ideal for plants and fish to coexist. To add detailed characterization of part BBa_K2447000 on phosphate concentrations specifically targeted for use in aquaponics systems, Lambert iGEM tested GFP expression of the phosphate sensor on extracellular phosphate levels from 0uM to 100uM in intervals of 20uM.
EXPERIMENTAL PROCEDURE
The characterization protocol began with the team’s biosensor cells being grown in chloramphenicol LB for 24 hours and later diluted to an OD600 value of 0.4. Then, the cells were pelleted and resuspended into MOPS media, which has minimal phosphate concentration relative to LB. To the 5 mL resuspension, the team added different phosphate concentrations between 0 to 100uM and waited 3 hours for GFP to be expressed. In order to measure the GFP expression, Lambert iGEM used a plate reader from Styczynski Research Group at Georgia Institute of Technology.
CHARACTERIZATION CURVE
Figure 4. Characterization curve showing the relationship between phosphate concentrations between 0 to 100uM and fluorescence/OD600 measured by a plate reader.
Figure 5. Prediction of relationship between GFP expression and phosphate concentrations ranging from 0 to 100uM made by deterministic ODE model.
Using data from the plate reader, Lambert iGEM created a characterization curve showing the relationship between phosphate concentrations and fluorescence/OD600. For phosphate concentrations ranging from 0 to 80uM, the decreasing trend in fluorescence/OD600 closely resembled Lambert iGEM’s ODE model prediction, shown in Figure 5. The fluorescence value for the 100uM phosphate concentration did not match the prediction from the model because the phosphate media was diluted improperly, causing its measured GFP expression to be higher than expected. Due to time constraints in the lab, the team was not able to conduct further testing and decided to use the characterization data for only 0 to 80uM of phosphate.
SAMPLE TESTING
Hypothetically, using the biosensor cells, aquaponics farmers should be able to identify the phosphate concentrations of unknown samples by utilizing the characterization curve. For instance, if the biosensor cells expressed Y arbitrary units of GFP, the user could find a point (X, Y) in the characterization curve in which X would be the corresponding phosphate concentration. This method would be applicable in identifying the phosphate concentration in unknown samples from real aquaponics systems such as a nutrient fertilizer mix, waste water from fish, and even tap water. Lambert iGEM did not have enough time to perform sample testing, but will be conducting experiments next year.
REFERENCES
[1] Santos-Beneit, F. (2015). The Pho regulon: a huge regulatory network in bacteria. Frontiers in Microbiology, 6. https://doi:10.3389/fmicb.2015.00402.
[2] 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.
[3] Storey, N. (2017, December 13). The Most Important Things To Know About Phosphorus. Retrieved October 03, 2020, from https://university.upstartfarmers.com/blog/most-important-things-about-phosphorus.
[4] Griffith, B. (2020). Phosphorus - Nutrient Management. Retrieved October 24, 2020, from https://www.cropnutrition.com/nutrient-management/phosphorus.
[5] Wanner, Barry. (1996). Signal transduction in the control of phosphate-regulated genes of Escherichia coli. Kidney international. 49. 964-7. 10.1038/ki.1996.136.
[6] 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.