Part:BBa_K2447000:Experience

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 above, is initiated once P_i (inorganic phosphate) molecules enter the cell by passing through PhoE porin proteins in the outer membrane. In the periplasmic space, P_i binds to the protein PstS, which carries P_i 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 P_i across the inner membrane. Different levels of P_i within the cytoplasm will then bind to the accessory protein PhoU and consequently activate or deactivate transcription of Pho Regulon genes.

Research has shown that higher levels of P_i in the cytoplasm deactivate the transcription of Pho Regulon genes [2]. When P_i is available in the cytoplasm, it binds to the accessory PhoU protein. The bound PhoU-P_i complex inhibits the PstB permease, preventing PstA/C from further transporting P_i into the cytoplasm. The same PhoU-P_i 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, P_i dissociates from PhoU - therefore restarting the cycle.

On the other hand, lower levels of P_i limit the accessory PhoU protein from binding to P_i; PhoU is therefore unable to inhibit the permease PstB. This allows P_i to enter the cytoplasm through the transmembrane channel PstA/C. Because of the initial lower levels of P_i, the PhoU-P_i complex is also unable to inhibit the histidine kinase PhoR. PhoR autophosphorylation occurs, and 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 downstream transcription of the Pho Regulon genes, while that of higher levels does not transcribe the regulatory genes.

Purpose of Characterization - Lambert_GA 2020

Figure 2: Diagram of part BBa_2447000 under different levels of extracellular inorganic phosphate.

As shown above, part BBa_K2447000 utilizes the Pho Regulon - replacing the Pho Regulon genes with Green Fluorescence Protein (GFP). Similar to the signaling pathway, under high extracellular phosphate levels, activation of the promoter via binding of phosphorylated PhoB transcription factor results in the downstream transcription and expression of GFP.

Lambert_GA 2020 used this part as a biosensor for hydroponics/aquaponics systems as research suggested the sensitivity range of the part is within the optimal range of phosphate concentration for hydroponics/aquaponics systems: 50uM to 100uM [3]. To improve upon the existing characterization of the phosphate sensor, Lambert iGEM tested with a greater number of phosphate concentrations ranging from 0uM to 100uM in intervals of 20uM.

Experimental Procedure - Lambert_GA 2020

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 100 micromolars and waited 3 hours for GFP to be expressed. In order to measure the GFP expression, Lambert_GA used a plate reader from Styczynski Research Group at Georgia Institute of Technology.

Results: Characterization Curve - Lambert_GA 2020

Figure 3: Characterization curve showing the relationship between phosphate concentrations between 0uM to 100uM and fluorescence/OD₆₀₀ measured by a plate reader.

Figure 4. Prediction of relationship between GFP expression and phosphate concentrations ranging from 0 to 100uM made by deterministic ODE model.

As shown in Figure 3, Lambert_GA 2020 created a characterization curve showing the relationship between phosphate concentrations and fluorescence/OD₆₀₀ using the data from the plate reader.

For phosphate concentrations ranging from 0uM to 80uM, the decreasing trend in fluorescence/OD₆₀₀ closely resembled the team's prediction in simulation of GFP expression from a deterministic ODE model, shown in Figure 4. 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 0uM to 80uM of phosphate.

References - Lambert_GA 2020

[1] Santos-Beneit, F. (2015). The Pho regulon: a huge regulatory network in bacteria. Frontiers in Microbiology, 6. 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). 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.

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